A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Summary

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent neointima for studies of restenosis.

Abstract

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

Introduction

In the era of expanded application of endovascular treatments for various cardiovascular diseases, restenosis after angioplasty is one of the major problems for patients undergoing such treatments. Damage to the vascular endothelium at the time of angioplasty, in concert with the atherosclerotic background, induces excessive smooth muscle cell proliferation in the medial layer, resulting in neointimal hyperplasia.^1,2 A viable animal model that recapitulates post-angioplasty neointimal hyperplasia is, thus, important not only for the investigation of disease mechanisms but also for the development of effective therapeutics to treat this pathology.

Protocol

NOTE: Ethics Statement: All procedures conform to the Guide for the Care and Use of Laboratory Animals (National Academies Press, 8th edition, 2011),  and protocols approved by the Institutional Animal Care and Use Committee at the University of Wisconsin. All surgeries were performed under isoflurane anesthesia (through inhaling, flow rate 2 ml/min), and all efforts were made to minimize suffering. Animals were euthanized in a chamber gradually filled with CO₂.

1. Induction of General Anesthesia and Skin Incision

1. Use male C57BL/6 mice with an age of 12–16 weeks and weight of 23–28 g. Anesthetize....

Results

Four weeks following surgery, harvest the wire-injured femoral artery and make a paraffin block specimen to analyze the neointima formation. Induce general anesthesia as described above. Open the chest immediately and insert a 20-gauge butterfly needle to the left ventricle. Connect the line to an infusion bottle. Perform perfusion fixation via drip infusion of PBS followed by 4 % paraformaldehyde (usually 10–20 ml, each). The infusion bottle is placed approximately 140 cm higher than the heart level so that the.......

Discussion

The wire injury procedure is applicable to all strains of mice as long as their anatomical structures are similar.⁴ In this paper, we used male C57BL/6 mice with an age of 12–16 weeks. As we have learned from our preliminary studies, the femoral arteries of mice younger than 10 weeks are often so small that wire insertion is quite challenging. On the other hand, wire insertion in mice older than 16 weeks is technically easier but tends to result in more variable neointimal hyperplasia. Female mice tend t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fixed Core Wire Guide	Cook	G02426	Diameter 0.015 inch, Straight
Dilation Forceps	Roboz	RS-4927	Curved, blunt tips
Dumont Tweesers #5	World Precision Instruments	14095	Straight, sharp tips
Dumont Vessel Cannulation Forceps	World Precision Instruments	503373
McPherson-Vannas Scissors	World Precision Instruments	501234
Mosquito Forceps	World Precision Instruments	501291
Ethilon Nylon Suture 9-0	Ethicon	7717G	9-0, Black Nylon Monofilament
Micro AROSuture, Sterile 11-0, 70 micrometers, MET Point	AROSurgical	VT4A00N07	11-0, Black Nylon Monofilament
3M Precise Multi-Shot DS Disposable Skin Stapler	3M	DS-25