Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Summary

The FeCl₃ induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Abstract

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

Introduction

The study of the mechanisms involved in the development of thrombosis and the evaluation of the effectiveness of anti-thrombotic drugs requires well established experimental animal models. Large animal models were the first to be used as they provide large vessels more similar to humans than rodents¹. However, high cost, the larger facilities required and the difficulty in manipulating them genetically are major drawbacks to their use and large animals are now limited to late preclinical studies once preliminary tests on rodents have given conclusive results². With wide availability of transgenic and knockout strains and their small size that min....

Protocol

All experiments involving animals were approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee (E/1534/2015/B). All surgical manipulations were performed under full anesthesia and the animals did not experience pain at any stage. All experiments described are non-recovery.

1. Preparation

1. Cut thin bands of filter paper (1 mm x 2 mm).
2. Freshly prepare 2 solutions of ferric chloride of 4% (w/v) and 6% (w/v) diluted into deionized water. Prepare rhodamine 6G solution 0.3% in PBS, filtered through 0.22 µm.
3. Cut a small piece (5 mm x 1 cm) from the white plastic of syringe wrapper.....

Results

The fluorescent intravital microscopy observation of the mesentery will reveal the accumulation of Rhodamine 6G labeled platelets and leukocytes along the vessel wall injured by FeCl₃. The progressive formation of a partial thrombus is monitored in a 200 µm mesentery vessel (Figure 1). A thrombus slowly appears and is clearly identifiable after the first minute of exposure to FeCl₃ (Figure 1, t = 60 sec). 40 sec after the removal of the filter paper soaked with FeCl.......

Discussion

The ferric chloride induced thrombosis model is an excellent research tool. As shown in this study, it is extremely easy to implement and when used in combination with intravital microscopy or Doppler flowmeter, it provides a good real-time monitoring of thrombus formation. Adjusting the time exposure and the concentration of FeCl₃, it also offers the possibility to produce either non-occlusive or occlusive thrombi.

However, this method also has some limitations. In the carotid arte.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Whatman chromatography paper	GE Healthcare	3030917
Iron (III) chloride 40% (w/v)	VWR	24212.298
Rhodamine 6G	Sigma	R4127
Inverted microscope	Olympus	IX81
Digital black-and-white camera	Olympus	XM10
Doppler flowmeter	Transonic	TS420
Nano-doppler flow probe	Transonic	0.5 PBS
Ketamine	Hospira	0409-2051-05
Xylazine (Rampun)	Bayer	75313
Petri dish	Sarstedt	82.1472
Insulin syringe (29 G)	BD Ultra-Fine	326103
Cotton tipped applicators	BSN medical	211827A
Dynek dysilk sutures	Dynek Pty Ltd	CS30100
Dulbecco's phosphate buffer saline (PBS)	Gibco life technologies	21600-069
Heating pad	Kirchner	T60