Ferric Chloride–Induced Arterial Thrombosis in Mice

Saravanan Subramaniam1, Sandip M. Kanse2

1 Justus Liebig University, Giessen, 2 University of Oslo, Oslo
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140140
Online Posting Date:  December, 2014
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Abstract

Because of its high sensitivity and reproducibility, the FeCl3‐induced arterial thrombosis mouse model is widely used to study molecular mechanisms as well as the efficacy of antithrombotic agents. The model also carries the additional advantages of being relatively easy to establish and requiring minimal instrumentation. However, the various parameters that influence the outcome must be optimized to the experimental requirements and the individual laboratory conditions. Here, we describe a standard protocol for FeCl3‐induced common carotid artery thrombosis, parameters that influence thrombus growth, ways to optimize the experiment, and interpretation of the data. © 2014 by John Wiley & Sons, Inc.

Keywords: carotid artery; ferric chloride; mouse; occlusion time; thrombosis; vascular injury

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Ferric Chloride–Induced Arterial Thrombosis in Mice
  • Basic Protocol 2: Harvesting of Samples for the Further Downstream Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Ferric Chloride–Induced Arterial Thrombosis in Mice

  Materials
  • Swiss Webster or C57BL/6J mice, 8 weeks old (Jackson Laboratory)
  • Anesthesia:
    • Isoflurane (Forane; Abbvie, cat. no. 6038497)/O 2or Cocktail A: 100 mg/ml ketamine/20 mg/ml xylazine stock in 0.9% NaCl or PBS
    • Cocktail B (MMF cocktail): 1 mg/ml Midazolame (Ratiopharm)/1 mg/ml Medetomidine (Pfizer)/0.5 mg/ml fentanyl (CuraMed Pharma GmbH) in 0.9% NaCl or PBS
  • Oxygen tank and regulator
  • Bepanthen—to avoid dryness of the eye (Bayer Vital GmbH, cat. no. 4245A)
  • Hair remover cream (SNÄ epil, Rufin Cosmetic, cat. no. 113401)
  • Antiseptic (Octeniderm, Schülke & Mayr GmbH, cat. no. 1224006)
  • 10% (617 mM) anhydrous ferric chloride (Fisher Scientific, cat. no. 02AC169430000)
  • Normal saline (0.9% NaCl; Braun, cat. no. 1343388205)
  • Isoflurane chamber (Vapor 19.3, Drägerwerk AG, Lübeck)
  • Syringe, 1 ml/2 ml (Injekt‐F, Braun, cat. no. 9166017V/4606027V)
  • Thermostat pad or heating pad (24V, 0 W, 300 × 150 × 12 mm, FMI Föhr Medical instruments GmbH, cat. no. 90039637)
  • Surgical tools (Fig.  ):
    • Dissection scissor, 4.0‐in. straight sharp, for cutting skin and soft tissue (Medicon, cat. no. 02.20.10)
    • Micro dissecting forceps, straight, fine sharp tips (Medicon, cat. no. 07.61.05)
    • Micro dissecting forceps, straight, blunt tips (Medicon, cat. no. 0207.55.10)
    • Micro dissecting forceps, serrated, full curve (Medicon, cat. no. 0207.61.25)
    • Iris scissor, straight (Medicon, cat. no. 0205.12.30)
    • Surgical suture (SOIE PERMA‐HAND, 7‐0, G‐6 8.0 mm 3/8c, sterile, 45 cm; Ethicon, cat. no. 02HAZ378)
    • Surgical tape (Leukofix, 1.25 cm × 9.2 cm, BSN Medical GmbH, cat. no. 0202136‐00AP)
  • Dissection/zoom stereo microscope (Olympus SZ1, cat. no. 1185354)
  • Sterile gauze (ES‐Kompressen, 100 m × 10 cm; Hartmann, cat. no. 1343388205)
  • For blood flow measurement:
    • Doppler flowmeter (Transonic TS420 Module)
    • Ultrasound Doppler transonic flow (T420 model 0.5 VB)
    • Chart recorder (Servogor 220, BBC Goerz)
    • Ultrasound transmission gel (Aquasonic 100)
  • Cotton swab—to remove excessive moisture from the vessel before ferric chloride application
  • Transparent sheet (soft plastic)—0.3 × 1.5 cm, to place below the carotid artery to avoid ferric chloride dilution
  • Whatman filter paper (1.5 × 1.5 mm pieces; circle, plain, sterile, MN60W, 150 mm) (Macherey‐Nagel, cat. no. 02202015)
  • Additional reagents and equipment for injection of mice (Donovan and Brown, 2006)

Basic Protocol 2: Harvesting of Samples for the Further Downstream Analysis

  Materials
  • Carotid artery thrombus–induced mice (see Basic Protocol 1)
  • Sodium citrate (Natrium citrate 3.13%, Eifelfango)
  • Phosphate‐buffered saline (PBS)
  • Tissue‐Tek (O.C.T compound, Sakura Finetek Europe B.V)
  • Liquid nitrogen (in Dewar flask)
  • Syringe, 2 ml (Injekt‐F; Braun, cat. no. 4606027V)
  • Surgical tools (Fig.  ):
    • Dissection scissor, 4.0‐in. straight sharp, for cutting skin and soft tissue (Medicon, cat. no. 02.20.10)
    • Micro dissecting forceps, straight, fine sharp tips (Medicon, cat. no. 07.61.05)
    • Micro dissecting forceps, straight, blunt tips (Medicon, cat. no. 0207.55.10)
    • Micro dissecting forceps, serrated, full curve (Medicon, cat. no. 0207.61.25)
    • Iris scissor, straight (Medicon, cat. no. 0205.12.30)
    • Surgical suture (SOIE PERMA‐HAND, 7‐0, G‐6 8.0 mm 3/8c, sterile, 45 cm; Ethicon, cat. no. 02HAZ378)
    • Surgical tape (Leukofix, 1.25 cm × 9.2 cm, BSN Medical GmbH, cat. no. 0202136‐00AP)
  • 2.0‐ and 0.5‐ml microcentrifuge tubes
  • Cryostat (Leica CM3050 S)
  • Microscope slides
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Figures

Videos

Literature Cited

Literature Cited
  Cooley, B.C. 2012. Murine models of thrombosis. Thrombosis Res. 129:S62‐S64.
  Denis, C., Methia, N., Frenette, P.S., Rayburn, H., Ullman‐Cullere, M., Hynes, R.O., and Wagner, D.D. 1998. A mouse model of severe von Willebrand disease: Defects in hemostasis and thrombosis. Proc. Natl. Acad. Sci. U.S.A. 95:9524‐9529.
  Denis, C.V., Dubois, C., Brass, L.F., Heemskerk, J.W., Lenting, P.J., and Biorheology Subcommittee of the SSC of the ISTH. 2011. Towards standardization of in vivo thrombosis studies in mice. J. Thromb. Haemostasis 9:1641‐1644.
  Eckly, A., Hechler, B., Freund, M., Zerr, M., Cazenave, J.P., Lanza, F., Mangin, P.H., and Gachet, C. 2011. Mechanisms underlying FeCl3‐induced arterial thrombosis. J. Thromb. Haemostasis 9:779‐789.
  Farrehi, P.M., Ozaki, C.K., Carmeliet, P., and Fay, W.P. 1998. Regulation of arterial thrombolysis by plasminogen activator inhibitor‐1 in mice. Circulation 97:1002‐1008.
  Fay, W.P., Parker, A.C., Ansari, M.N., Zheng, X., and Ginsburg, D. 1999. Vitronectin inhibits the thrombotic response to arterial injury in mice. Blood 93:1825‐1830.
  Kernan, W.N., Ovbiagele, B., Black, H.R., Bravata, D.M., Chimowitz, M.I., Ezekowitz, M.D., Fang, M.C., Fisher, M., Furie, K.L., Heck, D.V., Johnston, S.C., Kasner, S.E., Kittner, S.J., Mitchell, P.H., Rich, M.W., Richardson, D., Schwamm, L.H., Wilson, J.A., American Heart Association Stroke Council, Council on Cardiovascular and Stroke Nursing, Council on Clinical Cardiology, and Council on Peripheral Vascular Disease. 2014. Guidelines for the prevention of stroke in patients with stroke and transient ischemic attack: A guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 45:2160‐2236.
  Kurz, K.D., Main, B.W., and Sandusky, G.E. 1990. Rat model of arterial thrombosis induced by ferric chloride. Thrombosis Res. 60:269‐280.
  Lansberg, M.G., O'Donnell, M.J., Khatri, P., Lang, E.S., Nguyen‐Huynh, M.N., Schwartz, N.E., Sonnenberg, F.A., Schulman, S., Vandvik, P.O., Spencer, F.A., Alonso‐Coello, P., Guyatt, G.H., Akl, E.A., and American College of Chest, P. 2012. Antithrombotic and thrombolytic therapy for ischemic stroke: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed.: American College of Chest Physicians Evidence‐Based Clinical Practice Guidelines. Chest 141:e601S‐636S.
  Tseng, M.T., Dozier, A., Haribabu, B., and Graham, U.M. 2006. Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery. Thromb. Res. 118:275‐280.
  Van de Werf, F., Bax, J., Betriu, A., Blomstrom‐Lundqvist, C., Crea, F., Falk, V., Filippatos, G., Fox, K., Huber, K., Kastrati, A., Rosengren, A., Steg, P.G., Tubaro, M., Verheugt, F., Weidinger, F., Weis, M., and ESC Committee for Practice Guidelines. 2008. Management of acute myocardial infarction in patients presenting with persistent ST‐segment elevation: The Task Force on the Management of ST‐Segment Elevation Acute Myocardial Infarction of the European Society of Cardiology. Eur. Heart J. 29:2909‐2945.
  Wang, X. and Xu, L. 2005. An optimized murine model of ferric chloride‐induced arterial thrombosis for thrombosis research. Thrombosis Res. 115:95‐100.
  Westrick, R.J., Winn, M.E., and Eitzman, D.T. 2007. Murine models of vascular thrombosis (Eitzman series). Arterioscl. Thromb. Vasc. Biol. 27:2079‐2093.
  White, T.A., Pan, S., Witt, T.A., and Simari, R.D. 2010. Murine strain differences in hemostasis and thrombosis and tissue factor pathway inhibitor. Thrombosis Res. 125:84‐89.
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