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The present protocol describes a new technique of aortic banding in mice to induce pressure overload cardiac hypertrophy. For banding, a rubber ring with a fixed inner diameter is used. This new technique promises less variance and more reproducible data for future experiments.
Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of heart failure. The previously used technique is based on a threaded suture around the aortic arch tied over a blunted 27 G needle to create stenosis. This method depends on the surgeon manually tightening the thread and, thus, leads to high variance in the diameter size. A newly refined method described by Melleby et al. promises less variance and more reproducibility after surgery. The new technique, o-ring- aortic banding (ORAB), uses a non-slip rubber ring instead of a suture with a thread, resulting in reduced variation in pressure overload and reproducible phenotypes of cardiac hypertrophy. During surgery, the o-ring is placed between the brachiocephalic and left carotid arteries. Successful constriction is confirmed by echocardiography. After 1 day, correct placement of the ring results in an increased flow velocity in the transverse aorta over the o-ring-induced stenosis. After 2 weeks, impaired cardiac function is proven by decreased ejection fraction and increased wall thickness. Importantly, besides less variance in the diameter size, ORAB is associated with lower intra- and post-operative mortality rates compared with transverse aortic constriction (TAC). Thus, ORAB represents a superior method to the commonly used TAC surgery, resulting in more reproducible results and a possible reduction in the number of animals needed.
While physiological cardiac hypertrophy can be observed during development, exercise, and pregnancy, pathological cardiac hypertrophy responds to hemodynamic stress conditions like arterial hypertension, valvular heart disease, or gene mutations. Initially, the heart undergoes a remodeling characterized by increased cardiomyocyte size and thickening of the ventricular walls to maintain cardiac function1,2. On the other hand, pathological cardiac remodeling is associated with an increased risk for arrhythmia, sudden death, and high mortality. Finally, with time, it results in ventricular dilation, a strong decrease in contractile function, and eventual progression to heart failure (HF), which is associated with high morbidity, mortality, and societal costs3. Therefore, there is an urgent need to understand the molecular background in order to develop new therapeutic strategies4.
Aortic banding is a model that mimics pressure overload-induced left ventricular (LV) hypertrophy and heart failure in mice5. With this method, it is possible to examine the pathomechanisms of pressure overload-induced cardiac remodeling in vivo. The first aortic banding procedure in mice was reported by Rockman et al.6. Pressure overload is induced by a thread suture-based ligation around the aorta (between the brachiocephalic and left common carotid artery). To create a 0.4 mm diameter stenosis, a suture is placed around a 27 G needle and the aorta. After ligation, the needle is removed6,7.
Even though the needle diameter is fixed, the tightness of the thread is highly dependent on the surgeon and, therefore, affects the induced phenotype of cardiac hypertrophy. In addition, in the thread/suture-based method, there is a variable degree of stenosis diameter after surgery, associated with a high variance in mortality8,9. Moreover, training this method is challenging, especially regarding finding the right level and consistency in tightening the thread. Finally, at the beginning of training, high intra- and post-operative mortality due to disruption of the aorta or other tissue injury occurs, as well as high variation in the extent of stenosis in the surviving animals.
Recently, an optimized procedure of aortic banding was described by Melleby et al.10. They presented the ORAB (o-ring aortic banding) method with less variance in stenosis and highly reproducible levels of pressure overload by using a non-slip rubber o-ring with a fixed inner diameter of 0.71 mm, 0.66 mm, and 0.61 mm. In short, the o-ring is cut open, placed around the ascending arch, and closed again by threads. Other scientists using these o-rings reported less variability in the induced cardiac hypertrophy9. They also observed intra- and post-operative mortality, as well as better reproducibility and less variance in the induced hypertrophic phenotype9,11. The present article describes the procedure of this unique strategy in a step-by-step protocol. The expertise shared in this report will help other scientists to improve their techniques in this area.
To induce cardiac hypertrophy resulting in heart failure after 6 weeks, 12-week-old C57BL/6N male mice are recommended for surgery. A comparison 2 weeks after aortic banding between the mouse substrains C57BL/6N and C57BL/6J showed severe cardiac dysfunction and associated increased mortality in C57BL/6N mice. Therefore, these are better suited for models of heart failure12. Twelve-week-old male and female mice have an optimal size for exposure of the aorta and placement of the o-ring with special instruments.
The animal experiments were carried out under the principles of the regional committee (Ministerium für Energiewende, Landwirtschaft, Umwelt, Natur und Digitalisierung des Landes Schleswig-Holstein, permission number: V242-21249/2020 [38-4/20]). The mice used for the present study were obtained from a commercial source (see Table of Materials). The animals were kept under standard conditions with a 12 h light, 12 h night cycle; water and food were offered ad libitum.
1. Animal care
2. Preparation of the o-ring
NOTE: An o-ring with a fixed diameter of 0.4 mm is recommended to induce cardiac hypertrophy after 2 weeks. The extent and severity of the cardiac phenotype induced depend on the size of the o-ring diameter.
Figure 1: Performing the o-ring preparation for ligation. (A) An o-ring with a fixed diameter is cut with scissors or a scalpel on one side. (B) Image of an o-ring. (C) Each o-ring side is pierced with an 8-0 prolene thread. (D) O-ring pierced with two threads. (E) The threads of one ring side of the o-ring are pulled through the hole of the ligation aid. (F) Final position before placement: the threads of one side are placed through the hole of the ligation aid, while the threads of the other side are kept loose. Please click here to view a larger version of this figure.
3. Premedication of the mice and preparation of the operating field
4. Intubation of the mice
Figure 2: Intubation of the mouse. (A) Intubation instruments: (1) A 22 G i.v. cannula is used as an endotracheal tube (without mandarin); (2) Forceps; (3) Handmade laryngoscope (deformed/flattened cannula glued with wooden sticks and tape). (B) Performing intubation on the positioned heating pad. (C) Intubated mouse connected to a ventilator. Please click here to view a larger version of this figure.
5. Surgery and preparation for ring positioning
NOTE: Use sterile materials and instruments to avoid infections.
6. Ligation of the transverse aorta with the o-ring
Figure 3: Performing the o-ring implantation. (A) The aortic arch is exposed by three retractors of 1.0-2.5 mm. Both long threads of one ring side are passed under the aorta. (B) The o-ring will be placed by gently pushing the ring and pulling the threads. (C) The is o-ring is in the right position, and one cranial thread is knotted with caudal thread on each side. Please click here to view a larger version of this figure.
7. Suture and post-operative recovery
8. Confirmation of successful constriction and right position of the ring
Figure 4: Confirmation of transverse aorta ligation using pulse wave Doppler velocity measurement in the carotid arteries. (A) Representative pulsed wave Doppler velocity signals of the right carotid artery. (B) The stenosis results in a higher flow velocity in the right carotid artery than in the left. (C) The stenosis induced by constriction results in a flow velocity in the descending aorta of more than 2,400 mm/s. Sham mice show a flow velocity of 600-900 mm/s. Please click here to view a larger version of this figure.
Generally, aortic banding mimics human aortic stenosis and induces cardiac hypertrophy in mice. A successful procedure is characterized by heart tissue remodeling reflected by cardiac hypertrophy and reduced heart function5,6.
Directly 1 day after the operation, the effect of o-ring constriction of the transverse aorta can be determined in vivo by echocardiography13. Successful induction of aortic steno...
Thread/suture-based aortic banding has been used for many years to induce pressure-overload cardiac hypertrophy in mice. It is an established method to investigate the pathomechanisms of cardiac remodeling and disease progression in vivo. The limitations are the relatively high variance in the degree of stenosis and, consequently, the remodeling. The recently introduced ORAB technique first described by Melleby et al.10 optimizes the conventional method by using a rubber o-ring.
The authors have nothing to disclose.
This work was supported by the Bundesministerium für Bildung und Forschung (BMBF) to L.L., N.F., and O.J.M. (IVOLADMT-HF; FKZ 01KC2006A).
Name | Company | Catalog Number | Comments |
1 x long Fixators, 6 cm | 18200-01 | ||
2 x Blunt Retractors, 5 mm wide | 18200-11 | ||
2 x Short Fixators, 3 cm | 18200-02 | ||
2 x fine tip 45° angled forceps | FST (fine sience tools) | 11160-10 | |
3 x Blunt Retractors, 2.5 mm wide | 18200-10 | ||
3 x Retractor Wires | 18200-05 | ||
4-0 absorbable suture (Vicryl) | ETHICON | SABBKLT0 | Used to suture skin |
6-0 suture (Prolene), needle size:13 mm | ETHICON | JDP879 | Used to suture chest muscle |
8-0 suture (Prolene), needle size: 6.5 mm | ETHICON | RHBECH | Used to fast the Ring |
Anesthetizing Box, Small | Havard apparatus | 50-0108 | |
C57BL/6N mice | Charles River | ||
Fluosorber Activated Charcoal Filter Canister | Havard apparatus | 34-0415 | Used to induce and maintain anesthesia |
Handmade laryngsopcope | Intubationshelp | ||
Harvard Apparatus Anesthetic Vaporizer | Havard apparatus | Used to induce and maintain anesthesia | |
Heating pad + rectal probe (LSI Letica Scientific Instruments:Temperature control unit HB 101/2 ) | Panlab/ Havard apparatus | Used to control and maintain body temperature | |
i.v. cannula blue 22-gauge (Vasofix Braunüle 0.9 x 25 mm) | B/Braunsharing Expertise | 4268091B | intubation |
isofluran | Baxter | Anesthesia | |
Kodan (betadine solution) | Schülke | 20003960-A | Desinfection |
ligation aid | FST (fine sience tools) | 18062-12 | Used to perform liagtion with O-ring |
Microscope Lighting: Schott VisiLED Set MC1500/S80-55 (+ controller) | SCHOTT | Ligth | |
Microscope camera (Leica IC80 HD) | Leica | Used for visualiation operating field | |
MiniVac Complete Anesthesia Systems for small rodents | Havard apparatus | 75-0233 | Used to induce and maintain anesthesia |
Mouse Ventilator MiniVent Type 845 | Havard Apparatus | 73-0044 | Used to ventilation during surgery |
Needle holder | FST (fine sience tools) | TE-10804 | |
O-ring, non-slip rubber (0.0018 mm x 0.020 mm) | Apple Rubber Products | Liagtion of the aortic arch | |
Scissors | FST (fine sience tools) | 14040-09 | Used to cut the skin and threads |
Small Animal Retraction System (Kit for Animals up to 200 g) | FST (fine sience tools) | 18200-20 | |
Small Base Plate, 20 x 30 cm | 18200-03 | ||
Table intgerated with heating pad + rectal probe + ECG and transducer tripod | FujiFilm Visual Sonics Imaging System | Echocardiography | |
Temgesic (Buprenorphin) | Indivior UK Limited | 997.00.00 | Pain pre-medication |
three-way stop cock (blue) | |||
Tramal (Tamadol) | Grünental | Pain post-medication | |
transducer probe MS400 (Visual Sonics) | FujiFilm Visual Sonics Imaging System | Echocardiography | |
Ultrasound system with cardioligy package | FujiFilm Visual Sonics Imaging System | Echocardiography | |
Vannas Spring Scissors - 2.5 mm Cutting Edge | FST (fine sience tools) | 15000-08 | Used to cut intercostal chest muscle |
vet ointment | Bepanten | Used to prevent eyes from drying out |
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