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* These authors contributed equally
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.
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.
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....
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
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
0.9% Saline | Fisher Scientific | BP358-212 | NaCl used to make a solution of 0.9% saline |
1 mL Syringe | Becton, Dickinson and Company | 309659 | |
190 Proof Ethanol | KOPTEC | V1101 | Used to make a 70% ethanol solution to use for prepping the mouse for surgery |
2,2,2 Tribromoethanol | Sigma Aldrich | 48402 | |
25 Yard Black Braided Silk Suture (5-0) | DEKNATEL | 136082-1204 | |
26G x 3/8 Needle | Becton, Dickinson and Company | 305110 | |
2-methyl-2-butanol | Sigma Aldrich | 240486 | |
7.5 mL Transfer Pipet, Graduated to 3 mL | Globe Scientific Inc. | 135010 | |
Alcohol Prep Pads (70% Isopropyl Alcohol) | Medline | MDS090735 | |
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 long | World Precision Instrument | 15922-G | |
DMP-30 activator | Electron microscopy Services | 13600 | |
Dodenyl Succinic Anhydride/ DDSA | Electron microscopy Services | 13700 | |
Doggy Poo Bags/animal carcass disposal bag | Crown Products | PP-RB-200 | |
Doppler FlowProbe | Transonic 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 Tips | Integra Miltex | 18-784 | |
Filter Paper | VWR | 28310-106 | |
Fine Scissors - Sharp-Blunt | Fine Science Tools | 14028-10 | |
Finger Loop Ear Punches | Fine Science Tools | 24212-01 | |
Gauze Sponges 2” x 2” – 12 Ply | Dukal Corporation | 2128 | |
Glutaraldehyde (10% solution) | Electron microscopy Services | 16120 | |
Integra Miltex Carbon Steel Surgical Blade #10 | Integra® Miltex® | 4110 | |
Iron (III) Chloride | SIGMA-ALDRICH | 157740-100G | |
Knife Handle Miltex® Extra Fine Stainless Steel Size 3 | Integra Lifesciences | 157510 | |
L-aspartic acid | Sigma Fisher | A93100 | |
L-aspartic acid | Fisher Scientific | BP374-100 | |
Lead Nitrate | Fisher Scientific | L-62 | |
LEICA S8AP0 Microscope | LEICA | No longer available | No longer available from the company |
LEICA S8AP0 Microscope Stand | LEICA | 10447255 | No longer available from the company |
Light-Duty Tissue Wipers | VWR | 82003-822 | |
Micro Dissecting Forceps; 1x2 Teeth, Full Curve; 0.8 mm Tip Width; 4" Length | Roboz Surgical Instrument Company | RS-5157 | |
Osmium Tetroxide 4% aqueous solution | Electron microscopy Services | 19150 | |
Paraformaldehyde (16% solution) | Electron microscopy Services | 15710 | |
Potassium ferricyanide | SIGMA-ALDRICH | P-8131 | |
Propylene Oxide, ACS reagent | Electron microscopy Services | 20401 | |
Rainin Classic Pipette PR-10 | Rainin | 17008649 | |
Research Flowmeter | Transonic Systems Inc. | T402B01481 | Model: T402 |
Scotch Magic Invisible Tape, 3/4" x 1000", Clear | Scotch | 305289 | |
Small Animal Heated Pad | K&H Manufacturing Inc. | Model: HM10 | |
Sodium Cacodylate Buffer 0.2M, pH7.4 | Electron microscopy Services | 11623 | |
Sterile Cotton Tipped Applicators | Puritan Medical Products | 25-806 1WC | |
Steromaster Illuminator | Fisher Scientific | 12-562-21 | No longer available from the company |
Surgical Dumont #7 Forceps | Fine Science Tools | 11271-30 | |
Thiocarbohydrazide (TCH) | SIGMA-ALDRICH | 88535 | |
Universal Low Retention Pipet Tip Reloads (0.1-10 µL) | VWR | 76323-394 | |
Uranyl Acetate | Electron microscopy Services | 22400 | |
Veet Gel Cream Hair Remover | Reckitt Benckiser | 3116875 | |
White Antistatic Hexagonal Weigh Boats, Medium, 64 x 15 x 19 mm | Fisher Scientific | S38975 | |
WinDAQ/100 Software for Windows | DATAQ Instruments, Inc. | Version 3.38 | Freely available to download. https://www.dataq.com/products/windaq/ |
ZEISS AxioCam Icc 1 | ZEISS | 57615 |
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