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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

Abstract

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

Introduction

Thrombosis is the formation of a blood clot that partially or completely blocks a blood vessel, impeding the natural flow of the blood. This leads to severe and fatal cardiovascular events, such as ischemic heart disease and strokes. Cardiovascular diseases are the leading cause of morbidity and mortality, and cause one in four deaths worldwide1,2,3. Although thrombosis is manifested as a malfunction of the vascular system, it could be a result of an underlying microbial or viral infection, immune disorder, malignancy, or metabolic condition. The flow of blood is maintained b....

Protocol

All experiments discussed here were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky.

NOTE: Surgical instruments are listed in Figure 1 and the Table of Materials. C57BL/6J mice, 8-10 weeks old, male/female or relevant genetically manipulated (Knockout or Knockin) strains were used.

1. FeCl3-induced carotid artery injury

Representative Results

The data are generally presented as time to occlusion, or time required to form a fully occlusive thrombus. These data can be plotted as a Kaplan-Meier survival curve (Figure 4A)19, a dot plot with bars showing the terminal blood flow at the time of either cessation of the blood flow or the termination of an experiment (Figure 4B), or as a line graph (Figure 4C). Thrombus stability can be studied using this t.......

Discussion

The topical application of FeCl3 to the vasculature to induce thrombosis is a widely used technique, and has been instrumental in establishing roles for various platelet receptors, ligand signaling pathways, and their inhibitors20,21,22,23. The mechanism through which FeCl3 causes thrombosis is multifaceted; previously, endothelial denudation was considered a cause of thro.......

Acknowledgements

The authors thank the members of the Whiteheart Laboratory for their careful perusal of this manuscript. The work was supported by grants from the NIH, NHLBI (HL56652, HL138179, and HL150818), and a Department of Veterans Affairs Merit Award to S.W.W., R01 HL 155519 to B.S., and NIBIB intramural program grant to R.D.L.

....

Materials

NameCompanyCatalog NumberComments
0.9% Saline Fisher Scientific BP358-212NaCl used to make a solution of 0.9% saline 
1 mL Syringe Becton, Dickinson and Company 309659
190 Proof Ethanol KOPTECV1101 Used to make a 70% ethanol solution to use for prepping the mouse for surgery 
2,2,2 TribromoethanolSigma Aldrich48402
25 Yard Black Braided Silk Suture (5-0)DEKNATEL136082-1204
26G x 3/8 Needle Becton, Dickinson and Company 305110
2-methyl-2-butanolSigma Aldrich240486
7.5 mL Transfer Pipet, Graduated to 3 mLGlobe Scientific Inc.135010
Alcohol Prep Pads (70% Isopropyl Alcohol)MedlineMDS090735
Araldite GY 502 Electron microscopy Services 10900
Cell Culture Dish 35mm X 10mm Corning Incorporated 430165
Compact Scale Ward's Science 470314-390
Dissecting Scissors, 12.5 cm longWorld Precision Instrument15922-G
DMP-30 activator Electron microscopy Services 13600
Dodenyl Succinic Anhydride/ DDSAElectron microscopy Services 13700
Doggy Poo Bags/animal carcass disposal bagCrown Products PP-RB-200
Doppler FlowProbeTransonic Systems Inc.MA0.5PSB
EMBED 812 resin Electron microscopy Services 14900
Ethyl Alcohol, anhydrous 200 proof Electron microscopy Services 15055
Eye Dressing Forceps, 4" Full Curved, Standard, 0.8mm Wide TipsIntegra Miltex18-784
Filter Paper VWR28310-106
Fine Scissors - Sharp-BluntFine Science Tools 14028-10
Finger Loop Ear Punches Fine Science Tools 24212-01
Gauze Sponges 2” x 2” – 12 Ply Dukal Corporation2128
Glutaraldehyde (10% solution)Electron microscopy Services 16120
Integra Miltex Carbon Steel Surgical Blade #10Integra® Miltex®4110
Iron (III) Chloride SIGMA-ALDRICH157740-100G
Knife Handle Miltex® Extra Fine Stainless Steel Size 3Integra Lifesciences 157510
L-aspartic acidSigma Fisher A93100
L-aspartic acidFisher Scientific BP374-100
Lead Nitrate Fisher Scientific L-62
LEICA S8AP0 MicroscopeLEICANo longer availableNo longer available from the company
LEICA S8AP0 Microscope Stand LEICA10447255No longer available from the company
Light-Duty Tissue Wipers VWR82003-822
Micro Dissecting Forceps; 1x2 Teeth, Full Curve; 0.8 mm Tip Width; 4" LengthRoboz Surgical Instrument CompanyRS-5157
Osmium Tetroxide 4% aqueous solution Electron microscopy Services 19150
Paraformaldehyde (16% solution)Electron microscopy Services 15710
Potassium ferricyanideSIGMA-ALDRICHP-8131
Propylene Oxide, ACS reagent Electron microscopy Services 20401
Rainin Classic Pipette PR-10Rainin17008649
Research Flowmeter Transonic Systems Inc.T402B01481Model: T402
Scotch Magic Invisible Tape, 3/4" x 1000", ClearScotch 305289
Small Animal Heated PadK&H Manufacturing Inc.Model: HM10
Sodium Cacodylate Buffer 0.2M, pH7.4Electron microscopy Services 11623
Sterile Cotton Tipped Applicators Puritan Medical Products 25-806 1WC
Steromaster Illuminator Fisher Scientific 12-562-21No longer available from the company
Surgical Dumont #7 Forceps Fine Science Tools 11271-30
Thiocarbohydrazide (TCH)SIGMA-ALDRICH88535
Universal Low Retention Pipet Tip Reloads (0.1-10 µL)VWR76323-394
Uranyl AcetateElectron microscopy Services 22400
Veet Gel Cream Hair RemoverReckitt Benckiser3116875
White Antistatic Hexagonal Weigh Boats, Medium, 64 x 15 x 19 mmFisher Scientific S38975
WinDAQ/100 Software for WindowsDATAQ Instruments, Inc.Version 3.38Freely available to download. https://www.dataq.com/products/windaq/
ZEISS AxioCam Icc 1ZEISS57615

References

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