Here we describe a step-by-step protocol of surgical aorta debanding in the well-established mice model of aortic-constriction. This procedure not only allows studying the mechanisms underlying the left ventricular reverse remodeling and regression of hypertrophy but also to test novel therapeutic options that might accelerate myocardial recovery.
To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, mice undergo RR upon removal of the aortic constriction. In this paper, we describe a step-by-step procedure to perform a minimally invasive surgical aortic debanding in mice. Echocardiography was subsequently used to assess the degree of cardiac hypertrophy and dysfunction during LV remodeling and RR and to determine the best timing for aortic debanding. At the end of the protocol, terminal hemodynamic evaluation of the cardiac function was conducted, and samples were collected for histological studies. We showed that debanding is associated with surgical survival rates of 70-80%. Moreover, two weeks after debanding, the significant reduction of ventricular afterload triggers the regression of ventricular hypertrophy (~20%) and fibrosis (~26%), recovery of diastolic dysfunction as assessed by the normalization of left ventricular filling and end-diastolic pressures (E/e' and LVEDP). Aortic debanding is a useful experimental model to study LV RR in rodents. The extent of myocardial recovery is variable between subjects, therefore, mimicking the diversity of RR that occurs in the clinical context, such as aortic valve replacement. We conclude that the aortic banding/debanding model represents a valuable tool to unravel novel insights into the mechanisms of RR, namely the regression of cardiac hypertrophy and the recovery of diastolic dysfunction.
The constriction of the transverse or ascending aorta in the mouse is a widely used experimental model for pressure overload-induced cardiac hypertrophy, diastolic and systolic dysfunction and heart failure1,2,3,4. Aortic-constriction initially leads to compensated left ventricle (LV) concentric hypertrophy to normalize wall stress1. However, under certain circumstances, such as prolonged cardiac overload, this hypertrophy is insufficient to decrease the wall stress, triggering diastolic and systolic dysfunction (pathological hypertrophy)5. In parallel, changes in extracellular matrix (ECM) lead to the collagen deposition and crosslinking in a process known as fibrosis, which can be subdivided into replacement fibrosis and reactive fibrosis. Fibrosis is, in most of the cases, irreversible and compromises myocardial recovery after overload relief6,7. Nevertheless, recent cardiac magnetic resonance imaging studies revealed that reactive fibrosis is able to regress in the long term8. Altogether, fibrosis, hypertrophy and cardiac dysfunction are part of a process known as myocardial remodeling that rapidly progresses towards heart failure (HF).
Understanding the features of myocardial remodeling has become a major objective for limiting or reversing its progression, the latter known as reverse remodeling (RR). The term RR includes any myocardial alteration chronically reversed by a given intervention, such pharmacological therapy (e.g., antihypertensive medication), valve surgery (e.g., aortic stenosis) or ventricular assist devices (e.g., chronic HF). However, RR is often incomplete due to the prevailing hypertrophy or systolic/diastolic dysfunction. Thus, the clarification of RR underlying mechanisms and novel therapeutic strategies are still missing, which is mostly due to the impossibility to access and study human myocardial tissue during RR in most of these patients.
To overcome this limitation, rodent models have played a significant role in advancing our understanding of the signaling pathways involved in HF progression. Specifically, aortic debanding of mice with an aortic constriction represents a useful model to study the molecular mechanisms underlying adverse LV remodeling9 and RR10,11 as it allows the collection of myocardial samples at different time points in these two phases. Moreover, it provides an excellent experimental setting to test potential novel targets that can promote/accelerate RR. For instance, in aortic stenosis context, this model might provide information about the molecular mechanisms involved in the vast diversity of myocardial response underlying the (in)completeness of the RR6,12, as well as, the optimal timing for valve replacement, which represents a major shortcoming of the current knowledge. Indeed, the optimal timing for this intervention is a subject of debate, mainly because it is defined based on the magnitude of aortic gradients. Several studies advocate that this time point might be too late for the myocardial recovery as fibrosis and diastolic dysfunction are often already present12.
To our knowledge, this is the only animal model that recapitulates the process of both myocardial remodeling and RR taking place in conditions such as aortic stenosis or hypertension before and after valve replacement or the onset of anti-hypertensive medication, respectively.
Seeking to address the challenges summarized above, we describe a surgical animal model that can be implemented both in mice and rats, addressing the differences between these two species. We describe the main steps and details involved when carrying out these surgeries. Finally, we report the most significant changes taking place in the LV immediately before and throughout RR.
All animal experiments comply with the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85–23, revised 2011) and the Portuguese law on animal welfare (DL 129/92, DL 197/96; P 1131/97). The competent local authorities approved this experimental protocol (018833). Seven-week-old male C57B1/J6 mice were maintained in appropriate cages, with a regular 12/12 h light-dark cycle environment, a temperature of 22 °C and 60% humidity with access to water and a standard diet ad libitum.
1. Preparation of the surgical field
2. Mice preparation and intubation
3. Preparation for surgery (for both banding and debanding surgeries)
4. Ascending aortic banding surgery
NOTE: For a detailed protocol description, consult 2,3,4,13.
5. Post-operative care
6. Aortic debanding surgery
7. Echocardiography to assess cardiac function and left ventricular hypertrophy in vivo
8. Hemodynamic assessment
9. Aortic banding/debanding procedure in rats
Post-operative and late survival
The perioperative survival of the banding procedure is 80% and the mortality during the first month is typically <20%. As previously mentioned, the success of the debanding surgery is highly dependent on how invasive the previous surgery was. After a learning curve, the mortality rate during the debanding procedures is around 25%. For this mortality accounts mostly deaths during the surgery procedure, including aorta or left atrium rupture (in rats, the survival rate is higher in both surgical procedures).
Aortic banding and myocardial remodeling
The success of aortic constriction was verified by increased LV end-systolic pressure (LVESP) and by Doppler aortic flow velocities >2.5 m/s, which corresponds to a pressure gradient of 25 mmHg using the modified Bernoulli equation (Figure 5). Compared to SHAM mice, banding induced LV hypertrophy as assessed by increased LV mass (Table 1 and Figure 5) and impaired diastolic function evident by higher filling pressures (ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (E'), (E/e'), and left ventricular end-diastolic pressure (LVEDP) and prolonged relaxation (t, Table 1, Figure 5, and Figure 6) within 7 weeks. Ejection fraction was still preserved at this stage of the disease.
Histologically, seven-weeks of aortic banding induced significant cardiomyocyte hypertrophy and fibrosis (Figure 7).
Aortic debanding and myocardial reverse remodeling
In mice subjected to debanding, successful removal of aortic stenosis was verified by echo Doppler velocities (Table 1 and Figure 5). Overall, debanding promoted a significant decrease of afterload (decreased LVESP) and LV hypertrophy (assessed by morphometry, echocardiography, and histology). Moreover, we observed normalization of diastolic function and aortic velocities (Table 1, Figure 5, Figure 6, and Figure 7).
Table 1: Left ventricle morphofunctional changes assessed by echocardiography and by hemodynamics.
Critical steps | Advice |
Invasiveness of the banding surgery | It is important to avoid: ● prolonged occlusion of the ascending aorta during the ligation, which may lead to lung edema and activation of inflammatory pathways capable of influencing the phenotype and disease severity15 ● bleeding of the mammary artery which, if not timely circumvented, can lead to decreased blood pressure and promote higher amounts of fibrosis when re-opening the thorax (debanding); ● damaging mice pleura and lungs; Mini left lateral thoracotomy for banding and debanding (same place; present study) vs left lateral thoracotomy for the banding and a sternotomy for the debanding surgery11: ● the first is less invasive and have a short-recovery time, which improves the success of the open-chest haemodynamic performed two weeks later. Neverthless, the use of same position to re-open the chest can increase the number of complications due to adhesions (around left atrium, pulmonary artery, etc). Overcome this issue by having extracarefull during banding procedure. |
Suture internalization | Can be prevented by using: ● two banding sutures side-by-side16; ● silk instead of polypropylene11; ● titanium clips or a O-rings around the aorta to induce its constriction21; ● double loop-clip thecnique15; ● inflatable cuff to carry out supracoronary aortic banding22. |
Physiological parameters | During surgery it is important to monitor: ● heart rate; ● blood oxygenation, keeping it above 90% (specially during aorta manupilation); ● anesthesia, keeping it at the lowest dose possible without inflicting discomfort on the animal. |
Table 2: Critical steps of the protocol.
Figure 1: Ultra fine surgical instruments used for the banding and debanding procedures. (A) 2 needle-holders and a scalpel blade; 2 catheters for mice intubation and a scissor; a scalpel, 2 curved forceps, a ligation aid, a microsurgical scissor, 3 straight forceps; (B) and 26G-needle and blunted 26G-needle curved to fit the mice small thoracic opening properly. Please click here to view a larger version of this figure.
Figure 2: Aortic banding procedure. (A) The thoracic approach to the ascending aorta performed with the help of a magnetic fixator retraction system (3 retractors are visible). (B) The ascending aorta is clearly dissected and visible. The blunted needle and the polypropylene suture 6-0 are placed in the right position to perform the aortic banding. Please click here to view a larger version of this figure.
Figure 3: Aortic debanding procedure. (A) The mouse is placed in a magnetic retraction system, representing a handy tool to retract the muscles and tissues. The mouse is intubated for mechanical ventilation. A rectal probe controls temperature and an oximeter is placed on the right mice paw to monitor blood oxygenation during surgery. Fibrosis and adherent tissue is carefully removed around the aorta and suture, to be able to cut the suture (B) and (C). Please click here to view a larger version of this figure.
Figure 4: Experimental protocol design for mice. Myocardial remodeling (red) and reverse remodeling (green) are shown in the bottom together with all evaluation tasks. Of note, debanding surgery can give rise to two groups of animals with distinct degrees of reverse remodeling. Thus, we obtained DEB mouse with complete (DEB-COMP) and incomplete (DEB-INCOM) myocardial recovery. Please click here to view a larger version of this figure.
Figure 5: Echocardiographic assessment of cardiac structure and function. (A) Aortic flow velocities; (B) LV mass; (C) Ventricular dimensions (LV diameter, LVD) and wall thickness (LV posterior wall, LVPW and LV anterior wall, LVAW); (D) Transmitral flow (peak of pulse Doppler wave of late mitral flow velocity, A, and peak of pulsed Doppler wave of early mitral flow velocity, E) and (E) Myocardial velocities (late diastolic mitral annular tissue velocity, A'; early diastolic mitral annular tissue velocity, E' and systolic mitral annular tissue velocity, S'). Please click here to view a larger version of this figure.
Figure 6: Representative pressure-volume loops for SHAM, BA and DEB groups. Data were continuously acquired at 1000 Hz and subsequently analyzed off-line by PVAN software. Please click here to view a larger version of this figure.
Figure 7: Myocardial hypertrophy and fibrosis assessed histologically. (A) Left ventricle hypertrophy assessed by cardiomyocytes sectional area of hematoxylin-eosin (HE)-stained sections (5 µm) from SHAM (n = 17), BA (n = 14) and DEB group (n = 12). (B) Left Ventricular interstitial fibrosis and representative images of Red Sirius-stained sections (5 µm) from SHAM (n = 17), BA (n = 13) and DEB (n = 12). Please click here to view a larger version of this figure.
The model proposed herein mimics the process of LV remodeling and RR after aortic banding and debanding, respectively. Therefore, it represents an excellent experimental model to advance our knowledge on the molecular mechanisms involved in the adverse LV remodeling and to test novel therapeutic strategies able to induce myocardial recovery of these patients. This protocol details steps on how to create a rodent animal model of aortic banding and debanding with a minimally invasive and highly conservative surgical technique to reduce the surgical trauma.
The most critical step of the protocol is related to the degree of surgical aggression during aortic banding. The success of the subsequent aortic debanding surgery depends enormously on a minimally invasive banding procedure that avoids tissue aggression and fibrosis around the aorta and, therefore, a less-invasive approach is mandatory (Table 2). Suture internalization is associated with less LV hypertrophy and better cardiac function16 (Table 2) and makes the debanding procedure impossible to perform without causing an aortic rupture. In the present study, we tried to use silk, since it creates more scar tissue at the banding site, triggering a more stable degree of pressure overload. However, in our hands, the debanding surgery was more demanding when silk was used since it is a multifilament wire making it's total removal from the aorta more difficult. Nevertheless, these are technical issues that are widely protocol-and-operator-dependent, and these variations, type of suture, is not incompatible with good technical practices and reproductive results. Physiological parameters monitoring during banding and especially during debanding is mandatory for the success of the model implementation (Table 2).
In 1991, Rockman et al., described the transverse aorta constriction (TAC) in mouse for the first time4. Since then a considerable amount of papers came out providing numerous versions of this procedure with variations with respect to the animal age/size17, mice genetic background18, the diameter of the needle/constriction19, the material used for banding, the aortic location of the banding, the duration of the banding19 and debanding11. All these methodological alternatives are valid as long as they fulfill the aims of each study. However, we should stress out that the progression of the disease towards heart failure is faster and thus RR is more incomplete when selecting: 1) longer banding durations, 2) heavier/older the mice20 and 3) smaller needle diameter used for the aortic constriction (higher percentage of aortic constriction)16.
The duration of the banding and the debanding significantly impact the stage of the disease and, therefore, the recovery during RR. Likewise, choosing the right timing for debanding is mandatory to adjust to the severity of the disease envisaged. The results observed in our study are in accordance with pre-existence animal11,21 and human studies22, except for cardiomyocytes hypertrophy, where some studies showed its normalization10,21 and others its partial regression23. Moreover, studies have shown that, fibrosis regression can occur in the long term (70 months for human patients)24. The results seem to be dependent on the technique used to address fibrosis25. Recently, Treibel et al. were able to differentiate between cellular (myocytes, fibroblast, endothelial, red blood cells) and extracellular (ECM, blood plasma) compartments in patients with aortic stenosis after aortic valve replacemennt (AVR) using cardiovascular magnetic resonance with T1 mapping22. They described that regression of LV mass following AVR can be driven by 1) matrix regression alone, where the extracellular volume reduces; 2) cellular regression alone, where extracellular volume increases; 3) or by a proportional regression in cellular and matrix compartments, where the extracellular volume is unchanged22. These authors concluded that, following AVR, while diffuse fibrosis and myocardial cellular hypertrophy regress, focal fibrosis does not resolve. Thus, diffuse interstitial fibrosis, as assessed by matrix volume, is a potential therapeutic target. In our study, reduction of fibrosis seems to occur within 2 weeks of RR and before cardiomyocytes hypertrophy normalization. Also, sacrificing the animals 2 weeks after the debanding was the perfect timing to obtain ventricular diversity among DEB group, namely animals with diastolic dysfunction persistence (DEB-INCOM) and others with complete LV mass reversal and diastolic function improvement (DEB-COM). Moreover, as soon as 2 weeks after debanding, we have previously shown significant right ventricular changes in the banding group that partially recover after debanding26, while Bjornstad et al. reported normalization of fetal genes expression, indicative of myocardial remodeling within the same timeframe11.
The surgical procedure of banding/debanding can also be performed in rats26, however, some differences should be highlighted. Due to its bigger size, rats have more muscle layers than mice which decreases aortic visualization and hinders positioning the ligature around the aorta. On the other hand, the risk of damaging adjacent tissues and organs, such as atria or lungs, are minimized. To overcome the issue of suture internalization we used a larger polypropylene ligature in rats to hold tight the aorta (6.0 instead of 7.0 polypropylene).
Due to aorta manipulation, debanding surgery might decrease cardiac output by imposing additional afterload on LV and thus impair the circulatory and respiratory system. Compared to mice, rats seem to be more resistant to more extended anesthetic period and therefore are easier to keep the physiological respiratory parameters controlled during the long debanding surgery. In rats, LV hypertrophy development is faster than mice, but it takes longer to progress to heart failure. Thus, the debanding surgery can be done between 5-9 weeks after banding procedure without compromising ejection fraction26.
The major limitation of the banding/debanding animal model is the demanding microsurgical skills and technique of the operator, usually requiring a long learning curve to accomplish the debanding surgery. Another limitation is the impossibility to perform close chest hemodynamics in mouse and rat, which will be more physiologic. However, by using this method is obligatory to insert the catheter from the right carotid artery to LV which is, in this particular case not feasible since in banding animals ascending aorta is constricted before the carotid branches. Moreover, in mouse, we were not able to measure load-independent contractility (ESPVR) and diastolic parameters (slope of EDPVR) by performing vena cava occlusion maneuver, an important parameter for an adequate characterization of myocardial function. We found this maneuver difficult to perform in mice with ascending aorta constriction due to their small size (20-25g).
Future application of the banding/debanding animal model includes the development of novel therapeutic approaches to myocardial diseases and the characterization of the pathways that underlie the process of LV remodeling and RR.
In conclusion, this clinically-relevant model allows to temporally and mechanistically characterize the progression towards HF, as well as, its recovery since it allows the collection of myocardial samples in different stages of myocardial remodeling and RR. Moreover, it proves to be a useful experimental model for testing therapeutic strategies aimed at the recovery of the failing heart.
The authors have nothing to disclose.
The authors thank Portuguese Foundation for Science and Technology (FCT), European Union, Quadro de Referência Estratégico Nacional (QREN), Fundo Europeu de Desenvolvimento Regional (FEDER) and Programa Operacional Factores de Competitividade (COMPETE) for funding UnIC (UID/IC/00051/2013) research unit. This project is supported by FEDER through COMPETE 2020 – Programa Operacional Competitividade E Internacionalização (POCI), the project DOCNET (NORTE-01-0145-FEDER-000003), supported by Norte Portugal regional operational programme (NORTE 2020), under the Portugal 2020 partnership agreement, through the European Regional Development Fund (ERDF), the project NETDIAMOND (POCI-01-0145-FEDER-016385), supported by European Structural And Investment Funds, Lisbon’s regional operational program 2020. Daniela Miranda-Silva and Patrícia Rodrigues are funded by Fundação para a Ciência e Tecnologia (FCT) by fellowship grants (SFRH/BD/87556/2012 and SFRH/BD/96026/2013 respectively).
Absorption Spears | F.S.T | 18105-03 | To absorb fluids during the surgery |
Blades | F.S.T | 10011-00 | To perform the skin incision |
Buprenorphine | Buprelieve | Analgesia drug | |
Catutery | F.S.T | 18010-00 | To prevent exsanguination |
Catutery tips | F.S.T | 18010-01 | To prevent exsanguination |
cotton swab | Johnson's | To absorb fluids during the surgery | |
Depilatory cream | Veet | To delipate the animal | |
Disposable operating room table cover | MEDKINE | DYND4030SB | To cover the surgical area |
Echo probe | Siemens | Sequoia 15L8W | Ultrasound signal aquisition |
Echocardiograph | Siemens | Acuson Sequoia C512 | Ultrasound signal aquisition |
End-tidal CO2 monitor | Kent Scientific | CapnoStat | To control expiration gas saturation |
Forcep/Tweezers | F.S.T | 11255-20 | To dissect the tissues and aorta |
Forcep/Tweezers | F.S.T | 11272-30 | To dissect the tissues and aorta |
Forcep/Tweezers | F.S.T | 11151-10 | To dissect the tissues and aorta |
Forcep/Tweezers | F.S.T | 11152-10 | To dissect the tissues and aorta |
Gas system | Penlon Sigma Delta | To anesthesia and mechanical ventilation | |
Hemostats | F.S.T | 13010-12 | To hold the suture before tight the aorta |
Hemostats | F.S.T | 13011-12 | To hold the suture before tight the aorta |
Ligation aids | F.S.T | 18062-12 | To place a suture around the aorta |
Magnetic retractor | F.S.T | 18200-20 | To help keep the animal in a proper position |
Needle holder | F.S.T | 12503-15 | To suture the animal |
Needle 26G | B-BRAUN | 4665457 | To serve as a molde of aortic constriction diameter |
Oxygen | Air Liquide | To anesthesia and mechanical ventilation | |
Polipropilene suture | Vycril | W8304/W8597 | To suture the animal and to do the constriction |
Povidone-iodine solution | Betadine® | Skin antiseptic | |
PowerLab | Millar instruments | ML880 PowerLab 16/30 | PV loop Signal Aquisition |
Pulse oximeter | Kent Scientific | MouseStat | To control heart rate and blood saturation |
PVAN software | Millar Instruments | To analyse the haemodynamic data | |
PV loop cathether | Millar instruments | SPR-1035. 1.4 F | PV loop Signal Aquisition |
Retractor | F.S.T | 17000-01 | To provide a better overview of the aorta |
Scalpet handle | F.S.T | 10003-12 | To perform the skin incision |
Scissors | F.S.T | 15070-08 | To cut the suture in debanding surgery |
Scissors | F.S.T | 14084-09 | To cut other material during the surgery e.g. suture, papper |
Sevoflurane | Baxter | 533-CA2L9117 | |
Temperature control module | Kent Scientific | RightTemp | To control animal corporal temperature |
Ventilator | Kent Scientific | PhysioSuite | To ventilate the animal |
Water-bath | Thermo Scientific™ | TSGP02 | To maintain water temperature adequate to heat the P-V loop catethers |