This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.
Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological similarities to humans. Efforts have been dedicated to creating a model of pressure-overload induced heart failure, and ascending aortic banding while still supra-coronary and not a perfect mimic of aortic stenosis in humans, closely resembling the human condition.
The purpose of this study is to demonstrate a minimally invasive approach to induce left ventricular pressure overload by placing an aortic band, precisely calibrated with percutaneously introduced high-fidelity pressure sensors. This method represents a refinement of the surgical procedure (3Rs), resulting in homogenous trans-stenotic gradients and reduced intragroup variability. Additionally, it enables swift and uneventful animal recovery, leading to minimal mortality rates. Throughout the study, animals were followed for up to 2 months after surgery, employing transthoracic echocardiography and pressure-volume loop analysis. However, longer follow-up periods can be achieved if desired. This large animal model proves valuable for testing new drugs, particularly those targeting hypertrophy and the structural and functional alterations associated with left ventricular pressure overload.
Heart failure (HF) is a life-threatening disease that affects millions of people worldwide, causing major social and economic impacts1. One of its significant etiologies is aortic valve disease or aortic stenosis (AS). Aortic stenosis is more prevalent in advanced age and ranks as the second most common valvular lesion in the United States. AS-related mortality has also increased in Europe, particularly in countries without access to recent interventional procedures2. Given the complexity of HF and the scarcity of therapeutic innovations, there is a pressing need for reliable animal models that can replicate the human condition and facilitate the testing of new interventions3. While rodent models outnumber large animal models, the latter offers several advantages due to their size and physiological similarities, allowing the testing of drug doses and medical devices intended for human use.
The aim of this method is to establish a reproducible model of ascending aortic banding (AAB) applicable to most large animal species used in biomedical research. In this study, the procedure is demonstrated in swine using a minimally invasive approach, adhering to the 3Rs principles (replacement, reduction, and refinement4). This approach ensures the creation of an accurate pressure gradient, resulting in high reproducibility (potentially reducing the number of required animals). Additionally, the small surgical incision (2-3 cm) minimizes surgical insult, improving animal well-being compared to more aggressive approaches like sternotomy and larger thoracotomies5 (refinement). Furthermore, providing a video demonstration of the method, along with detailed descriptions in the literature, could potentially reduce the need for animals used solely for training purposes (replacement), further decreasing animal usage. This model can be adapted for different swine strains/breeds with distinct growth rates and induces sustained pressure overload, leading to significant hypertrophy after 1 or 2 months of follow-up.
Current methods employ fixed stenosis6, disregarding animal size variability, or calculate gradient using fluid-filled pressure readings7, which are less reliable than high-fidelity pressure sensors and are susceptible to signal damping8. Another approach uses a single pressure measurement distal to the stenosis5. However, calibrating the stenosis through simultaneous proximal and distal pressure signals using percutaneously delivered high-fidelity pressure sensors represents a substantial optimization of the protocol, resulting in improved group homogeneity. By visually demonstrating this method, other researchers should be able to replicate it without significant obstacles, increasing the availability of this model while promoting the application of the 3Rs principles.
The animal experiments were performed at the Experimental Surgery laboratory in the University of Porto, Cardiovascular Research and Development Centre (UnIC, Porto, Portugal). The institutional animal ethics committee approved the study in accordance with the National Authority for Animal Health (Direcção-Geral de Alimentação e Veterinária, DGAV, Ref: 2021-07-30 011706 0421/000/000/2021). The experimenters were either licensed (FELASA-equivalent Laboratory Animal Sciences authorization) or were cardiothoracic surgeons or anesthesiologists. Animals used in this work were males from a Landrace x Pietrain background and were acquired from a breeder licensed by DGAV (PTAH03). Starting weight of the animals was 20-25 kg, which allowed for a maximum of 2-month follow-up (70-80 kg, Figure 1). Longer follow periods are compromised due to the significant animal growth, which our infrastructures were unable to handle.
1. Anesthesia and vital sign monitoring
2. Arterial cannulation
3. Ascending aortic banding (preparation)
4. Left ventricle (LV)/Aorta catheterization
5. Ascending aortic banding (constriction)
During the initial development of the model, the mortality rate was approximately 30%, with animals dying from acute heart failure after banding and surgical complications. However, after the model was established, surgical complications became less common, and the mortality rate dropped to around 15%. The two deaths that occurred were due to aortic rupture during dissection.
The use of high-fidelity pressure sensors enables obtaining high-quality pressure signals (Figure 2), allowing real-time and accurate calibration of the stenosis. This ensures that all operated animals experience a similar degree of left ventricular pressure overload, reducing variability within the group. Moreover, the catheter itself has a 2.3 F shaft, which has minimal impact on flow obstruction compared to larger fluid-filled catheters. After an initial investment, the catheters can be reused multiple times, and if sterilization is needed, ethylene oxide can be used (usually available through collaboration with surgical departments at the hospital).
The trans-stenotic gradient can be calculated in real-time by the software, which measures the pressure difference between the left ventricle (proximal pressure) and the distal aorta (distal pressure). A few minutes of stabilization between each constriction step ensures that the left ventricle has time to adapt. After determining the desired constriction degree, a 15 min stabilization period should be applied to ensure that the banding degree remains stable and the animal is compensated (Figure 2A).
This approach is superior to other methodologies that do not measure trans-stenotic gradient in real-time and lack both the homogeneity of having a similar gradient between all animals (92.3 ± 2.3 mmHg, mean and standard error of the mean, respectively, for 7 operated animals) and tight monitoring of left ventricular pressures. Additionally, this approach avoids the difficulties associated with performing transthoracic echocardiography in swine, particularly in certain breeds like the Vietnamese potbellied pig, which has a more significantly protruding sternum.
Transthoracic echocardiography can confirm aortic banding both immediately after the surgery and during follow-up time points (Figure 3). The banding surgery results in significant stenosis of the aorta with turbulent flow, which can be qualitatively evaluated or quantified using continuous wave Doppler. Figure 2 displays representative images of 2-month follow-up echocardiography, showing significant aortic stenosis (upper row) and left ventricular concentric hypertrophy (middle and bottom rows). Two months after banding, the animals develop significant cardiac hypertrophy. The macroscopic evaluation revealed larger hearts and a thicker left ventricular wall (Figure 4). The two-month follow-up period was determined based on the growth rate of the used animals, as a longer follow-up period would result in animals too large to be handled by our infrastructures.
Figure 1: Schematics of the aortic banding protocol. After receiving 20-25 kg male pigs, the animals are submitted to a 1 week quarantine period. On the day of the procedure, the animals are anesthetized, the LV and aorta are catheterized, and high-fidelity pressure sensors are placed, followed by aortic banding and animal recovery. The whole procedure, once mastered, lasts around 2 h. Two months after surgery, the animals are submitted to a terminal evaluation, including collection of samples and measurement of physiological variables. AB-aortic banding, Ao-aorta, LV-left ventricle, PV-pressure-volume, RHC-right heart catheterization, US-ultrasound. Please click here to view a larger version of this figure.
Figure 2: Pressure measurements during aortic banding. (A) Representative traces of LV and aortic (distal to the banding) pressures during aortic banding. Zoom in on LV and aortic pressure before (B) and after (C) constriction, showing the gradient creation (difference between peak systolic LV and aortic pressure). (D) Pull-off of the ventricular pressure sensor, transitioning from the aorta proximal to the banding to the aorta distal to the banding. AP-arterial pressure, LVP-left ventricle pressure, MC-high-fidelity pressure sensor. Please click here to view a larger version of this figure.
Figure 3: Transthoracic echocardiography. Follow-up at 2 months after surgery reveals significant stenosis of the aorta (black arrow, upper row). LV hypertrophy is apparent, both in 2D (white arrows, middle row), as well as in M-mode, which also demonstrates concentric hypertrophy (white arrows, bottom row). The vertical bar corresponds to 3 cm, and 2D PSAX images were acquired at a depth of 15 cm. Please click here to view a larger version of this figure.
Figure 4: Post-mortem macroscopic analysis of the heart. Aortic banding leads to cardiomegaly, with clear hypertrophy of the LV wall. Heart slices are base, mid-cavity, and apex from left to right. Pericardial adhesions can be seen throughout the epicardium. Scale bars represent 1 cm (upper row) and 4 cm (lower row). Please click here to view a larger version of this figure.
In recent years, several studies have utilized surgical aortic banding as a model for left ventricular pressure overload and heart failure (descending9 to the ascending aorta10), allowing researchers to obtain various phenotypes tailored to their specific needs. Although using such models requires costly equipment and specialized knowledge, the information they provide is invaluable. Swine, due to its size and similarity to the human heart, serves as an ideal model11, gaining ethical acceptance as organ donors for xenotransplantation.
The main critical step in this method is the dissection of the aorta and the placement of the banding material (nylon cable or ePTFE graft) around it. During this step, several complications can occur, including laceration or rupture of the surrounding structures or the aorta itself. Controlling such complications can be achieved by placing a pursue-string suture or a mattress suture with pledgets on the hole if bleeding can be controlled to properly visualize the wound. It is strongly recommended to have the procedure performed by a cardiothoracic surgeon, which significantly reduces complication and mortality rates.
Another critical step is the constriction of the aorta, which should be done in sequential steps with stabilization periods in between. Paying close attention to systemic peripheral pressures is crucial, as sustained significant hypotension (mean arterial pressure below 60 mmHg) may result from the LV’s inability to cope with the current stenosis. If not resolved, especially when ventricular pressures start to drop as well, acute heart failure will lead to the loss of the animal. The removal of the nylon cable or titanium clip is necessary when hypotension does not spontaneously resolve.
However, the main limitation of this model, and many aortic banding models, is the band’s location relative to the coronary ostia. Supra-coronary banding placement does not entirely mimic aortic stenosis and may lead to increased blood pressure in the coronary circulation, which might be protective12. Limited evidence suggests no differences between sub-coronary and supra-coronary aortic banding in pigs13, indicating that the increased complications associated with sub-coronary banding surgery may not be worthwhile.
Depending on the animal strain used and the follow-up time, band internalization may become an issue. Although mainly described in rodents14, it has also been observed in the pulmonary artery of pigs15. Using ePTFE graft segments significantly increases the contact area and eliminates the occurrence of band internalization. However, ePTFE grafts are more expensive, and when using slow-growing breeds, such as the Vietnamese pot-bellied pig, band internalization is not an issue when using nylon zip ties. Researchers should choose their approach based on the animal breed used.
For fast-growing breeds, long-term follow-up might be challenging due to animal size (availability of infrastructure and equipment large enough to handle >100 kg animals) and prohibitive maintenance costs.
Another limitation of this model, as well as all models requiring pericardial space access, is the presence of significant pericardial adhesions after surgery. Our experience shows no difference between closing or not closing the pericardial incision after band placement. While it does not affect function, dissecting the heart and identifying different structures becomes more time-consuming, and the epicardium is likely to be damaged if the pericardium is fully separated.
This minimally invasive method represents a significant refinement of the typical surgical procedure, leading to an uneventful and speedier recovery. The use of two high-fidelity catheters for simultaneous pressure measurement and real-time gradient measurement significantly improves the accuracy of the procedure and the reproducibility of the model, leading to a reduction in the number of animals required. The model can be applied to the study of new therapeutic interventions or devices aimed at left ventricular hypertrophy, as well as the determination of new pathophysiological mechanisms associated with left ventricular pressure overload.
The authors have nothing to disclose.
This work was supported and funded under the QREN project 2013/30196, the "la Caixa" Banking Foundation, the Fundação para a Ciência e Tecnologia (FCT) project, LCF/PR/HP17/52190002. JS and EB were supported by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 813716. PdCM was supported by the Stichting Life Sciences Health (LSH)-TKI project MEDIATOR (LSHM 21016).
3-0 PDS II suture | Ethicon | Z683G | Aorta banding |
5-0 prolene | Ethicon | 7472H | Aorta banding |
ACUSON NX2 Ultrasound System | Siemens | (240)11284381 | Vascular Access and Echocardiography |
Arterial Extension 200 cm | PMH | 303.0666 | Anesthesia Maintenance |
Atlan A300 Ventilator | Draeger | 8621300 | Ventilation |
Bone cutters | Fehling | AMP 367.00 | Aorta banding |
Cefazolin 1000 mg | Labesfal | 100063 | Antibiotic |
Chlorhexidine 4% Wash Solution | AGA | 19110008 | Cleaning |
Doyen Intestinal Forceps | Aesculap | EA121R | Intubation |
Echogenic Introducer Needle | Teleflex | AN-04318 | Vascular Access |
Endotracheal tube | Intersurgical | 8040070 | Intubation |
ePTFE vascular graft (5 mm x 40 cm) | GORE-TEX | S0504 | Aorta banding |
Extension line 100 cm | PMH | 303.0394 | Anesthesia Induction |
F.O. Laryngoscope | Luxamed | E1.317.012 | Intubation |
F.O. Miller Blade 4 204 x 17 mm | Luxamed | 3 | Intubation |
Fenestrated Sterile Drape | Bastos Viegas | 4882-256 | Aseptic Technique |
Fentanyl 0.5 mg/10 mL | B.Braun | 5758883 | Anesthesia / Analgesia |
Guidewire 260 cm J-tip | B.Braun | J3 FC-FS 260-035 | Left Ventricle catheterization |
Infusomat Space Infusion Pump | B.Braun | 24101800 | Fluids / Drug administration |
Intercostal retractor | Fehling Surgical | MRP-1 | Thoracotomy |
Introcan Certo IV Catheter 20G | B.Braun | 4251326 | Fluids / Drug administration |
Isotonic Saline Solution 0.9% | B.Braun | 5/44929/1/0918 | Fluids / Drug administration |
Ketamidor 100 mg/mL | Richter pharma | 1121908AB | Anesthesia Induction |
L10-5v Linear Transducer | Siemens | 11284481 | Vascular Access |
Midazolam 15 mg/3 mL | Labesfal | PLB762-POR/2 | Anesthesia Induction |
Mikro-cath | Millar | 63405(1) | Pressure recording |
MP1 guide catheter 6 Fr | Cordis | 67027000 | Left Ventricle catheterization |
Needle Holder | Fehling Surgical | ZYY-5 | Aorta banding |
Non-woven adhesive | Bastos Viegas | 442-002 | Fluids / Drug administration |
P4-2 Phased Array Transducer | Siemens | 11284467 | Echocardiography |
Perfusor Compact Syringe Perfusion Pump | B.Braun | 8717030 | Fluids / Drug administration |
Pressure Signal Conditioner | ADinstruments | PCU-2000 | Pressure recording |
Propofol Lipuro 2% | B.Braun | 357410 | Anesthesia Maintenance |
Radifocus Introducer II Standard Kit B – Introducer Sheath | Terumo | RS+B60K10MQ | Vascular Access |
Radiopaque marker | Scanlan | 1001-83 | Aorta banding |
Scissors | Fehling Surgical | Thoracotomy | |
Skinprep (Chlorhexidine 2% / 70% Isopropyl alcohol) | Vygon | SKPC015ES | Disinfection |
Stopcock manifold (3 ports) | PMH | 310.0489 | Fluids / Drug administration |
Straight forceps | Fehling Surgical | ZYY-1 | Thoracotomy |
Stresnil 40 mg/mL | ecuphar | 572184.2 | Anesthesia Induction |
Syringe Luer Lock 20 cc | Omnifix B.Braun | 4617207V | Anesthesia Induction |
Syringe Luer Lock 50 cc | Omnifix B.Braun | 4617509F | Anesthesia Maintenance |
Transdermal fentanyl Patch 50 mcg/h | Mylan | 5022153 | Analgesia |
Ultravist | Bayer | KT0B019 | Angiography |
Universal Hemostasis Valve Adapter | Merit Medical | UHVA08 | Left Ventricle catheterization |
Velcro Limb Immobilizer | PMH | SU-211 | Animal stabilization |
Venofix A, 21 G | B.Braun | 4056337 | Anesthesia Induction |
Vista 120S Patient Monitor | Draeger | MS32997 | Monitoring |
Weck titanium clip | Teleflex | 523760 | Aorta banding |
Weck titanium clip applier | Teleflex | 523166 | Aorta banding |
Zhiem Vision | Iberdata | N/A | Fluoroscopy |