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Representative Results






Ferric Chloride-induced Murine Thrombosis Models

Published: September 5th, 2016



1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, 2Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 3Department of Pharmacology, Case Western Reserve University, 4Department of Biomedical Engineering, Case Western Reserve University

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi that occur in the arterial circulation stem from inappropriate platelet activation, aggregation and subsequent coagulatory mechanisms, and are implicated in heart attacks, strokes and extremity loss. The vessel wall is a complex system that includes multiple cell types and is influenced by a multitude of extrinsic factors including shear stress, circulating blood cells, hormones and cytokines, as well as expression of antioxidant proteins in....

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All procedures and manipulations of animals have been approved by Institutional Animal Care and Use Committees (IACUC) of The Cleveland Clinic in accordance with the United States Public Health Service Policy on the Humane Care and Use of Animals, and the NIH Guide for the Care and Use of Laboratory Animals.

1. Preparations:

  1. Fluorescent Dye for Labeling Platelets
    1. Prepare rhodamine 6G solution, 0.5 mg/ml, in saline and sterilize the solution with 0.22 µ.......

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Carotid Artery Thrombosis Model
In mice with C57BL6 background, we recommend using 7.5% FeCl3 to treat the vessel for 1 min as a starting point. Under treatment of 7.5% FeCl3, borders of the injured area and normal vessel wall are easily identified under microscope (See online video 1), suggesting that the endothelial layer was significantly damaged. The thrombi formed immediately upon FeCl3 treatment, and are observ.......

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The FeCl3-induced model is one of the most widely used thrombosis models, which can not only provide valuable information about genetic modifications on platelet function and thrombosis7,8,16,19,31-33, but can also be a valuable tool for evaluation of therapeutic compounds and strategies for treatment and prevention of atherothrombotic diseases11,17,34-37. Here we have shown our modifications and refinements of this model and showed additional evidence of the utility of this technique, wh.......

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This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta), R01 HL129179 (PI: Sen Gupta, Co-I: Li) and R01 HL098217 (PI: Nieman). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


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Name Company Catalog Number Comments
Surgical Scissors - Tungsten Carbide Fine Science Tools  14502-14 cut and hold skin
Micro-Adson Forceps - Serrated/Straight/12cm Fine Science Tools  11018-12 cut and hold skin
Metzenbaum Fino Scissors - Tungsten Carbide/Curved/Blunt-Blunt/14.5cm Fine Science Tools  14519-14   to dissect and separate soft tissue
Ultra Fine Hemostat - Smooth/Curved/12.5cm Fine Science Tools  13021-12 to dissect and separate soft tissue
Graefe Forceps - Serrated/Straight/10cm Fine Science Tools  11050-10 to dissect and separate soft tissue
Dumont #5 Fine Forceps - Biology Tips/Straight/Inox/11cm Fine Science Tools  11254-20  Isolate vessel from surounding tissue
Dumont #5XL Forceps - Standard Tips/Straight/Inox/15cm Fine Science Tools  11253-10 Isolate vessel from surounding tissue
Blunt Hook- 12cm/0.3mm Tip Diameter Fine Science Tools  10062-12 Isolate vessel from surounding tissue
Castroviejo Micro Needle Holders Fine Science Tools  12061-02 Needle holders
Suture Thread 4-0 Fine Science Tools  18020-40 For fix the incisors to the plate
Suture Thread 6-0 Fine Science Tools  18020-60 For all surgery and ligation
Kalt Suture Needles Fine Science Tools  12050-03
rhodamine 6G  Sigma 83697-1G To lebel platelets
FeCl3 (Anhydrous) Sigma 12321 To induce vessel injury
Papaverine hydrochloride Sigma P3510 To inhibit gut peristalsis.
Medline Surgical Instrument Sterilization Steam Autoclave Tapes Medline 111625 To fix the mouse to the plate
Fisherbrand™ Syringe Filters - Sterile 0.22µm Fisher 09-720-004 For sterlization of solutions injected to mice
Fisherbrand™ Syringe Filters - Sterile 0.45µm Fisher 09-719D To filter the FeCl3 solution
Sterile Alcohol Prep Pad Fisher 06-669-62 To sterilize the surgical site
Agarose  BioExpress E-3120-500 To make gel stage
Leica DMLFS fluorescent microscope Leica Intravital microscope
GIBRALTAR Platform and X-Y Stage System npi electronic GmbH
Streampix version 3.17.2 software NorPix

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