Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

Summary

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Abstract

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

Introduction

Arterial stenosis in coronary and peripheral arteries has a large effect on the morbidity and mortality of patients¹. One underlying pathological mechanism is myointima hyperplasia (MH), which is characterized by increased proliferation, migration, and synthesis of extracellular matrix proteins from vascular smooth muscle cells (SMC)². SMC are located in the media layer of the vessel and migrate upon stimulation to the surface of the lumen. Stimulatory signals include growth factors, cytokines, cell-cell contact, lipids, extracellular matrix components, and mechanical shear and stretch forces³

Protocol

Animals received humane care in compliance with the Guide for the Principles of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, and published by the National Institutes of Health. All animal protocols were approved by the responsible local authority (''Amt für Gesundheit und Verbraucherschutz, Hansestadt (Office for Health and Consumer Protection) Hamburg'').

1. Catheter Preparation

NOTE: Refer to the Table of Materials for information regarding the catheter.

1. Take catheter from the catheter holder.
2. Pull the guidewire from th....

Results

Balloon denudation is a suitable model to study the development of MH in mice. Animals recover well from the surgery and show an excellent physical condition post-operation. We established this model in 50 mice with less than 3% death rate due to the surgical procedure. Figures 1B-C show the key surgical steps. After a skin incision along the linea alba, identify the aorta abdominalis. Place microsurgical clamps (

Discussion

This article demonstrates a murine model to study the development of myointimal hyperplasia and allows the exploration of the underlying pathological processes and the testing of new drugs or therapeutic options.

The most critical step in this protocol is the denudation of the aorta. Special care should be paid during this step as excessive denudation will lead to aneurysm formation and model failure. On the other hand, if denudation is performed insufficiently, too little myointima will devel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-0 Ethilon suture	Ethicon	2814G
3 mL Syringe	BD Medical	309658
37% HCl	Sigma-Aldrich	H1758
5-0 prolene suture	Ethicon	EH7229H
6-0 prolene suture	Ethicon	8706H
Acid Fuchsin	Sigma-Aldrich	F8129-25G	Trichrome staining
Antigen retrieval solution	Dako	S1699
Azophloxin	Waldeck	1B-103	Trichrome staining
Bepanthen Eye and Nose ointment	Bayer	1578675	Eye ointment
Betadine Solution	Betadine Purdue Pharma	NDC:67618-152
C57BL/6J	Charles River	Stock number 000664
Clamp applicator	Fine Science Tools	18056-14	JAW DIMS: 4 x 0.75 mm LENGTH: 13 mm
Collagen 3	abcam	ab7778	Antibody
DAPI	Thermo Fischer	D1306
Donkey anti-Goat IgG AF555	Invitrogen	A21432	Secondary antibody
Donkey anti-Rabbit IgG AF488	Invitrogen	A21206	Secondary antibody
Donkey anti-Rabbit IgG AF488	Invitrogen	A11055	Secondary antibody
Donkey anti-Rabbit IgG AF555	Invitrogen	A31572	Secondary antibody
Ethanol 70%	Th. Geyer	2270
Ethanol 96%	Th. Geyer	2295
Ethanol absolute	Th. Geyer	2246
FAP	abcam	ab28246	Antibody
Forceps fine	Fine Science Tools	11251-20
Forceps standard	Fine Science Tools	11023-10
Glacial Acetic Acid	Sigma-Aldrich	537020
Hair clipper	WAHL	8786-451A ARCO SE
Heparin	Rotexmedica	PZN 3862340	25.000 I.E./mL
High temperature cautery kit	Bovie	18010-00
Image-iT FX Signal Enhancer	Invitrogen	I36933	Blocking solution
Light Green SF	Waldeck	1B-211	Trichrome staining
Microsurgical clamp	Fine Science Tools	18055-04	Micro-Serrefine - 4mm
MINI TREK Coronary Dilatation Catheter 1.20 mm x 6 mm / Rapid-Exchange	Abbott	1012268-06U
Molybdatophosphoric acid hydrate	Merck	1.00532.0100	Trichrome staining
NaCl 0,9%	B.Braun	PZN 06063042 Art. Nr.: 3570160
Needle holder	Fine Science Tools	12075-14
Novaminsulfon	Ratiopharm	PZN 03530402	Metamizole
Orange G	Waldeck	1B-221	Trichrome staining
Paraffin	Leica biosystems	REF 39602004
PBS pH 7,4	Gibco	10010023
PFA 4%	Electron Microscopy Sciences	#157135S
Ponceau S solution	Serva Electrophoresis	33427	Trichrome staining
Primary antibody diluent	Dako	S3022
Prolong Gold Mounting solution	Thermo Fischer	P36930	Mounting solution for immunofluorescence stained slides
Replaceable Fine Tip	Bovie	H101
Resorcin-Fuchsin Weigert	Waldeck	2E-30	Trichrome staining
Rimadyl	Pfizer	400684.00.00	Carprofen
Scissors	Fine Science Tools	14028-10
Scissors Vannas-style	Fine Science Tools	15000-03
Secondary antibody diluent	Dako	S0809
Fast acting Adhesive MINIS 3x1g	UHU	45370	Cyanoacrylate
Slide Rack	Ted Pella	21057
SM22	abcam	ab10135	Antibody
SMA	abcam	ab21027	Antibody
Staining dish	Ted Pella	21075
Surgical microscope	Leica	M651
Tabotamp fibrillar	Ethicon	431962	Absorbable hemostat
Transpore Surgical Tape	3M	1527-1
U-100 Insulin syringe	BD Medical	324825
Vessel Dilator	Fine Science Tools	18603-14
Vitro-Clud	Langenbrinck	04-0001
Weigerts iron hematoxylin Kit	Merck	1.15973.0002	Trichrome staining
Xylene	Th. Geyer	3410