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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 decr....

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 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.

Representative Results

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.......

Discussion

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.

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
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)ETHICONSABBKLT0Used to suture skin
6-0 suture (Prolene), needle size:13 mmETHICONJDP879Used to suture chest muscle
8-0 suture (Prolene), needle size: 6.5 mmETHICONRHBECHUsed to fast the Ring 
Anesthetizing Box, SmallHavard apparatus 50-0108
C57BL/6N miceCharles River
Fluosorber Activated Charcoal Filter CanisterHavard apparatus 34-0415Used to induce and maintain anesthesia
Handmade laryngsopcope Intubationshelp
Harvard Apparatus Anesthetic VaporizerHavard 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 4268091Bintubation 
isofluranBaxterAnesthesia
Kodan (betadine solution)Schülke20003960-ADesinfection 
ligation aidFST (fine sience tools)18062-12Used to perform liagtion with O-ring 
Microscope Lighting: Schott VisiLED Set MC1500/S80-55 (+ controller)SCHOTTLigth
Microscope camera (Leica IC80 HD)LeicaUsed for visualiation operating field 
MiniVac Complete Anesthesia Systems for small rodentsHavard apparatus 75-0233Used to induce and maintain anesthesia
Mouse Ventilator MiniVent Type 845Havard Apparatus 73-0044Used 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
ScissorsFST (fine sience tools)14040-09Used 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 tripodFujiFilm Visual Sonics Imaging SystemEchocardiography 
Temgesic (Buprenorphin)Indivior UK Limited997.00.00Pain pre-medication 
three-way stop cock (blue)
Tramal (Tamadol)GrünentalPain post-medication 
transducer probe MS400 (Visual Sonics) FujiFilm Visual Sonics Imaging SystemEchocardiography 
Ultrasound system with cardioligy package FujiFilm Visual Sonics Imaging SystemEchocardiography 
Vannas Spring Scissors - 2.5 mm Cutting EdgeFST (fine sience tools)15000-08Used to cut intercostal chest muscle 
vet ointment BepantenUsed to prevent eyes from drying out

References

  1. Frey, N., Olson, E. N. Cardiac hypertrophy: The good, the bad, and the ugly. Annual Review of Physiology. 65, 45-79 (2003).
  2. Bui, A. L., Horwich, T. B., Fonarow, G. C. Epidemiology and risk profile of heart failure. Nat....

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