O-Ring Aortic Banding Versus Traditional Transverse

Aortic Constriction for Modeling Pressure Overload-

Induced Cardiac Hypertrophy
Nesrin Schmiedel1, Anca Remes1, Mohsen Valadan2, Susanne Hille1, Andrea Matzen1, Derk Frank1, Norbert Frey2, Lorenz
Lehmann2, Oliver J. Müller1
1 2
Department of Internal Medicine III, German Centre for Cardiovascular Research, University Hospital Kiel, Partner Site Hamburg/Kiel/Lübeck Internal
Medicine III, German Centre for Cardiovascular Research, University Hospital Heidelberg, Partner Site Heidelberg/Mannheim

Corresponding Authors
Abstract
Nesrin Schmiedel
Nesrin.Schmiedel@uksh.de
Aortic banding in mice is one of the most commonly used experimental models for
Oliver J. Müller
cardiac pressure overload-induced cardiac hypertrophy and the induction of heart
Oliver.Mueller@uksh.de
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
Citation
the surgeon manually tightening the thread and, thus, leads to high variance in the
Schmiedel, N., Remes, A., Valadan, M.,
Hille, S., Matzen, A., Frank, D., Frey, N., diameter size. A newly refined method described by Melleby et al. promises less
Lehmann, L., Müller, O.J. O-Ring
variance and more reproducibility after surgery. The new technique, o-ring- aortic
Aortic Banding Versus Traditional
Transverse Aortic Constriction for banding (ORAB), uses a non-slip rubber ring instead of a suture with a thread, resulting
Modeling Pressure Overload-Induced
in reduced variation in pressure overload and reproducible phenotypes of cardiac
Cardiac Hypertrophy. J. Vis. Exp. (188),
e64455, doi:10.3791/64455 (2022). hypertrophy. During surgery, the o-ring is placed between the brachiocephalic and left
carotid arteries. Successful constriction is confirmed by echocardiography. After 1 day,
Date Published
correct placement of the ring results in an increased flow velocity in the transverse
October 6, 2022
aorta over the o-ring-induced stenosis. After 2 weeks, impaired cardiac function is
proven by decreased ejection fraction and increased wall thickness. Importantly,
DOI
besides less variance in the diameter size, ORAB is associated with lower intra-
10.3791/64455
and post-operative mortality rates compared with transverse aortic constriction (TAC).
URL Thus, ORAB represents a superior method to the commonly used TAC surgery,

jove.com/video/64455 resulting in more reproducible results and a possible reduction in the number of
animals needed.

Introduction

While physiological cardiac hypertrophy can be observed cardiac hypertrophy responds to hemodynamic stress
during development, exercise, and pregnancy, pathological conditions like arterial hypertension, valvular heart disease,

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com October 2022 • 188 • e64455 • Page 1 of 12
 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
Copyright © 2022 JoVE Journal of Visualized Experiments jove.com
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.
Protocol
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
October 2022 • 188 • e64455 • Page 2 of 12
 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
1. House the mice in specialized cages with bedding,
nesting material, a hiding place, and proper access to
drinking water and food.
2. Keep the animals under continuous
veterinary control and treatment.
NOTE: For mice ordered by external suppliers, please
assure 7 days of acclimation before starting the
procedure.
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.
Copyright © 2022 JoVE Journal of Visualized Experiments
1. First, perform one cut of the o-ring (see Table of
Materials) under the microscope using scissors or a
scalpel to enable the placement around the aorta (Figure
1A, B).
2. Pierce each ring side close to the cut with a needle
connected with an 8-0 non-absorbable suture and pull
the thread. Cut and leave 2-3 cm on one side and 2 cm
on the other side to fasten the o-ring around the aorta in
the final step (Figure 1C, D).
specialized
3. Before surgery, take the ligation aid (species instrument,
see Table of Materials) and pull the end of the thread
(which is kept longer) of one ring side through the hole of
the constriction (Figure 1E, F). Put the ligation aid with
the attached o-ring aside for placement in the following
step (step 6).
4. For the disinfection of the ring with the threads, place the
ring in an alcohol solution for half an hour. Afterward, lay
it on cellulose to dry. Keep the dried ring in a closed tub or
case until use. During the surgery, after pulling the thread
through the ligation aid, place the ring on a clean surface
until use.
jove.com October 2022 • 188 • e64455 • Page 3 of 12
 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.

2. After premedication, anesthetize the mouse in an

3. Premedication of the mice and preparation of
the operating field induction chamber with 2%-4% isoflurane mixed with
0.5-1.0 L/min of 100% O2.
1. To receive sufficient analgesia during the surgery, inject
3. Shave the fur on the left thorax side of the sedated
the analgesic buprenorphine (0.1 mg/kg, see Table of
mouse. After shaving, put the mouse back into the
Materials) intraperitoneally 20 min before proceeding to
isoflurane-filled chamber and wait for sufficient sedation
the surgery.
before intubating the animal.
NOTE: For the present study, the pain medication was
NOTE: The right time point of the sedation shows slow
used following the recommendations of the Gesellschaft
breathing but avoids snap breathing. Depending on the
für Versuchstierkunde/Society of Laboratory Animal
isoflurane gas setting, it takes 2-3 min to achieve the right
Science (GV-SOLAS).
level of sedation.

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 4. Turn the heating pad on before the surgery to maintain 7. Use this hand also to introduce the endotracheal tube into
the animal's body temperature (37 °C). Connect the the trachea. On the other hand, still hold the intubation
heating pad to a rectal probe (see Table of Materials) to aid. For intubation, use a 22 G cannula (see Table of
maintain the mouse's body temperature automatically. Materials) (Figure 2A[1]).

8. Connect the endotracheal tube position via a three-way

4. Intubation of the mice
stopcock to a ventilator (see Table of Materials) for
1. Prepare the required instruments (Figure 2A). mice to confirm the correct endotracheal tube position
Disinfect the laryngoscope before the surgery by placing (endotracheal).
it in alcohol for about 1-3 minutes and then keeping it for
9. Monitor the right ventilation according to the
drying overnight.
manufacturer's instructions (tidal volume of 200 µL and
2. Stretch a rubber band around the heating pad to fix the respiration rate between 100-150 breaths/min) (Figure
mouse with the front teeth on the plate. Place the sedated 2C).
mouse on the heating pad in a supine position.
10. Confirm sufficient anesthetic depth by a toe pinch reflex
3. Place the rubber band over the animal's front teeth to check (no reflex response).
extend the neck on the plate.
11. Turn the anesthesia setting to 2% isoflurane mixed with
4. Focus a light source on the throat for good visibility of 0.5-1.0 L/min 100% O2.
the opening of the trachea for the endotracheal intubation
12. Apply ophthalmic ointment on the eyes to avoid dryness
(Figure 2B).
during the surgery.
5. Open the mouth gently with one hand positioning an
13. Using a cotton swab, disinfect the surgery area 3 times
intubation aid (handmade laryngoscope, see Table of
with a commercially available disinfectant solution (see
Materials) (Figure 2A[3]).
Table of Materials).
6. With the other hand and small forceps, gently move the
tongue to clear the opening of the trachea.

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 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.

4. To expose the aortic arch, try to lift up and gently

5. Surgery and preparation for ring positioning
NOTE: Use sterile materials and instruments to avoid
infections.
1. Use scissors to make a 0.5-1 cm long skin incision in
the middle of a line between the xyphoid process and left
axilla. Use forceps to separate the muscle layer from the
underlying ribs and place two retractors (5 mm length,
see Table of Materials) into the incision to expose the
rib cage.
2. To start the left thoracotomy, perform a small incision
(~1-2 mm) in the intercostal muscles between the second
and third rib using micro spring scissors. Open the
thoracic cavity and spread the incision with 45° angled
forceps.
3. Place three chest retractors (1.0-2.5 mm length) into
the incision for opening the thoracic cavity to improve
visualization.
separate the thymus and fat tissue from the arch with fine
tip 45° angled forceps.
6. Ligation of the transverse aorta with the o-ring
1. Expose the aortic arch with 45° angled forceps in one
hand. Position, with the other hand, the o-ring connected
with the ligation aid via the threads of one side (step 2).
2. Pass the threads using the ligation aid under the aortic
arch from the caudal side to the cranial side of the
transverse aorta between the brachiocephalic and left
common carotid arteries (Figure 3A).
3. Take both threads between the ligation aid and the aortic
arch with the forceps carefully. Retract and remove the
ligation aid and gently position the o-ring around the arch
by pulling the threads on each side (Figure 3B).
4. After successful positioning, fix the o-ring with the threads
and a surgical knot. Make an additional one to avoid
opening the knot on each side (Figure 3C).

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 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.

Lay the mouse on the left side under the heat lamp in its
7. Suture and post-operative recovery
care unit and observe it until it is completely awake.

1. Remove the three chest retractors (2.5 mm length) from 7. Do not leave an animal unattended until it has regained
the incision. sufficient consciousness.

2. If needed, eliminate the residual air from the thorax by NOTE: An animal that has undergone surgery should get

filling it with a warm 0.9% isotonic salt solution. its own care unit (cage) for better recovery.

3. To expose the thorax incision for suture, take two 8. Perform pain management with tramadol (1 mg/mL) in

retractors (5 mm length) again to hold the skin on the drinking water for 7 days and buprenorphine (0.1 mg/

side. kg, 3x daily) by intraperitoneal injection for 3 days after

surgery if needed.
4. Close the thorax with two or three 6-0 non-absorbable
NOTE: Follow local animal ethics committee
sutures (see Table of Materials) and pinch off the
recommendations for postoperative analgesia.
outflow of the ventilator for 2 s to reinflate the lungs.
9. Check medication by weighing the water bottles daily and
5. Remove the two retractors and close the skin with three
watch the animal's behavior.
to five 4-0- absorbable sutures.

6. Turn off the isoflurane and monitor. When the animal

starts self-breathing, the whiskers are moving, and the
toe pinch reflexes can be triggered, extubate the mouse.

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 this measurement, position the transducer head
8. Confirmation of successful constriction and
parasternally on the right thorax side to localize the aortic
right position of the ring
arch by two-dimensional (2D) imaging ("B-mode").
1. One day after surgery, verify the stenosis using
1. Use the color Doppler to visualize the blood flow in
ultrasound by measuring the maximal flow velocity over
the aorta and measure with the pulsed wave Doppler
the stenosis.
blood flow velocity over the stenosis.
2. For measurements, use echocardiography with an NOTE: Sham-operated mice (control surgery
ultrasound system and a transducer probe with a without a constriction) show a blood flow velocity of
frequency of 30 MHz (see Table of Materials). ~600-900 mm/s. In addition, a successful ORAB also

3. As described above, maintain anesthesia using a mask results in an increased velocity flow ratio between

at 1.5%-2% isoflurane with 0.5-1.0 L/min of 100% O2. the right carotid (~150 mm/s) (RC, Figure 4A) and
the left carotid (~300 mm/s) LC, Figure 4B) in the
4. Place the anesthetized animal on the heating pad in a
mouse.
supine position. Connect the heating pad with a rectal
7. Visualize the right and left arteria carotis interna by
probe to maintain the body temperature at 37 °C ± 1 °C,
two-dimensional (2D) imaging (B-mode). Position the
and monitor the heart rate with an ECG using four mouse
transducer head horizontal on the left and right sides of
paw sensors (see Table of Materials).
the neck at a 45° angle and use the pulsed wave Doppler
5. For better visualization, use depilation creme.
to determine the blood flow velocity.
6. A successfully performed ORAB results in an NOTE: In sham-operated mice, the velocity flow in both
increased flow velocity over the stenosis as measured arteries is similar.
by ultrasound (~2,400 mm/s) (Figure 4C). For

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 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.

vivo by echocardiography13 . Successful induction of aortic

Representative Results stenosis is shown by increased blood flow velocity over the

Generally, aortic banding mimics human aortic stenosis and stenosis in the aorta measured by pulsed wave Doppler

induces cardiac hypertrophy in mice. A successful procedure ultrasound (Figure 4C). Additionally, as described above, we

is characterized by heart tissue remodeling reflected by determine the blood flow velocity ratio between the right and

cardiac hypertrophy and reduced heart function5 , 6 . left internal carotid arteries as a functional marker of ORAB
(Figure 4A,B).
Directly 1 day after the operation, the effect of o-ring
constriction of the transverse aorta can be determined in

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 Two weeks after placing the 0.4 mm o-ring, the successful
induction of cardiac hypertrophy can be determined by
echocardiography. The two-dimensional (2D) time-motion
mode (M-mode) displays the visualized cardiac structures
as they change over the cardiac cycle. Two weeks post
surgery, increased left ventricular (LV) posterior wall (LVPW)
and interventricular septum (IVS) diameters in diastole can be
observed (Figure 5B). Additionally, a progressive worsening
of cardiac function can be documented by measuring
decreased left ventricular ejection fraction (LVEF).

Figure 5: Confirmation of induced cardiac hypertrophy after 2 weeks post surgery by echocardiography. (A)
Representative M-mode image of a sham-operated mouse 2 weeks post surgery. (B) M-mode imaging of an ORAB-
operated mouse 2 weeks post surgery with increased left ventricular posterior wall (LVPW) and interventricular septum (IVS)
diameters in diastole. Please click here to view a larger version of this figure.

Discussion surgery. Another advantage is the faster learning and easier

placement in this method. With the thread/suture-based
Thread/suture-based aortic banding has been used for
process, finding the right degree of ligation around the aorta
many years to induce pressure-overload cardiac hypertrophy
usually takes time and experience. In contrast, this is not
in mice. It is an established method to investigate
necessary to the same extent with the o-ring approach
the pathomechanisms of cardiac remodeling and disease
because of the fixed diameter or the o-ring.
progression in vivo. The limitations are the relatively high
variance in the degree of stenosis and, consequently, the Open chest surgery is a critical point in the ORAB technique,
remodeling. The recently introduced ORAB technique first which is painful for the animal but necessary for o-ring ligation.
described by Melleby et al.10 optimizes the conventional In order to achieve better recovery and avoid the occurrence
method by using a rubber o-ring. of atelectasis, it is important to prevent lung injury and to
monitor ventilation through the ventilator. For the thread/
The most valuable benefit of this technique is the fixed
suture-based method, there is the opportunity for a minimally
diameter of the o-ring, which leads to less variability
invasive technique. In this procedure, the tissue is spread
in the degree of the stenosis and, consequently, to
with one pair of forceps only for putting the thread around the
more reproducible phenotypes of cardiac hypertrophy post

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 aorta7 , 13 . However, the disadvantage of this procedure is still
the possible injury of the aorta due to the ligation.
Recently, Nakao et al.9 reported a similar technique to the
ORAB approach. The so-called o-ring-induced transverse
aortic constriction (OTAC) is performed with a mini-
stereotomy without intubation. Similar to the present study,
the results show high reproducibility. However, sternotomy in
mice might be more painful for animals than spreading the
tissue with retractors.
The unique ORAB technique is an improvement to the thread/
suture-based technique. With the fixed diameter of the o-ring,
the variability of stenosis and subsequent cardiac hypertrophy
is lower, resulting in more reproducible results. Furthermore,
training in the procedure requires fewer mice. The low
mortality rate of this method suggests that it is a potentially
superior alternative to the thread/suture TAC model.
Disclosures
The authors have nothing to disclose.
Acknowledgments
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).
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