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.

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
<ol>
	<l...

<ol>
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The authors have no conflicts of interest related to this study.
ORCID profiles: S.J.: 0000-0001-6925-2116; S.W.W.: 00000-0001-5577-0473.

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...

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 by the complex interaction among diverse components of the vascular system, including endothelial cells, red/white blood cells, platelets, and coagulation factors4. Upon vascular injury, platelets interact with adhesive proteins on the subendothelial matrix and release their granular contents, which recruit more platelets5. Concurrently, the coagulation cascade is activated, leading to fibrin formation and deposition. Ultimately, a clot is formed, containing platelets and red blood cells trapped within a fibrin mesh6. Although antiplatelet and anticoagulant drugs are available to modulate thrombosis, spurious bleeding remains a major concern with these therapies, requiring fine-tuning of the dosages and combinations of these drugs. Thus, there is still an urgent need to discover new anti-thrombotic drugs7.
Thrombosis is studied using multiple methods to inflict vascular injury: mechanical (vessel ligation), thermal (laser injury), and chemical injury (FeCl3/Rose Bengal application). The nature of thrombosis varies depending on the location (arterial vs. venous), method, or extent of the injury. Among all these types, FeCl3-induced vascular injury is the most widely used method. It has been employed in mice, rats, rabbits, guinea pigs, and dogs8,9,10,11,12. The method is relatively simple, easy to use, and if major parameters are standardized, it is sensitive and reproducible in various vascular systems (e.g., arteries [carotid and femoral], veins [jugular], and arterioles [cremaster and mesenteric]) (Supplemental Table 1).
This model can also be used to further our understanding of the mechanics and morphology of clot formation. This technique uniquely offers the advantage of stopping thrombosis at various flow rate points, to study the intermediate stages of the process before it becomes occlusive. Recent advances in thrombosis research have used this model to focus attention on non-pharmacological methods of thrombolysis13 or non-invasive delivery of anti-thrombotic and/or fibrinolytic agents14,15. Several groups have shown that, when platelet membranes are coated with these therapeutics, the drugs can be activated upon thermal stimulation to target clots16. The techniques described here can be useful to such studies as validation of their findings at the single platelet level. In this manuscript, Protocol 1 describes the basic FeCl3-mediated vascular injury procedure, while Protocol 2 describes the method to collect and fix the vascular injury sample for further analysis by electron microscopy.

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ferric chloride-induced arterial thrombosis and sample collection for 3d 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.

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...

Watch this Scientific Journal Video about Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis at JoVE.com

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

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 ...

With this method, one can determine what components of platelets and the hemostatic system are important for thrombosis. Additionally, this method can determine how thrombus growth and architecture are affected at the cellular level. The main advantage of this technique is that it allows a reproducible way to study many stages of thrombosis on a structural level.A person new to this technique should avoid over-handling the tissue in the surgical area. This can lead to bleeding and can overcomplicate the identification of the carotid artery. Begin marking the injury site after exposing the carotid artery by loosely encircling the region between two surgical threads having a 0.1 millimeter size.Place the probe under the artery distal from the lower thread. Insert the plastic piece under the artery between the two threads to mark the site for iron chloride injury. Take flow measurements before performing injury to note the basal flow.Perform the injury by placing iron chloride-soaked filter paper on the artery for three minutes. Remove the filter paper after three minutes to prevent the variable extent of the injury and to facilitate flow counting by the probe. Add saline at the injury site to remove any excess residual iron chloride.Monitor the flow reading. When the flow drops below 50%of the initial value, remove the probe. Quickly dry the area and add the fixative to fix the injury area externally.Next, promptly hold the artery near the injury area with forceps. Cut downstream of the lower thread and upstream of the upper thread. Put the tissue on the plastic tissue culture dish in the same orientation as it was collected and add a few drops of the fixative.Using a scalpel, clean the extra tissue around the artery. Ensure recording the orientation of the blood flow by cutting one end horizontally and the other tapering or oblique. Place the sample in fixative for one hour at room temperature, then store the sample in 1%paraformaldehyde at 4 degrees Celsius until sending it down wet ice for electron microscopy analysis.The data was plotted as a Kaplan-Meier survival curve, 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, or as a line graph. The blood flow increased suddenly in some cases following a gradual decrease for a few minutes indicating partial shedding of the growing thrombus and was considered an embolization event. Occlusive thrombus morphology was studied using this method as well.The prompt collection of the sample during the thrombosis process is the most important step since delay in this process may lead to misinterpretation of our structural analysis.

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1.5K Views.  University of Kentucky. With this method, one can determine what components of platelets and the hemostatic system are important for thrombosis. Additionally, this method can determine how thrombus growth and architecture are affected at the cellular level. The main advantage of this technique is that it allows a reproducible way to study many stages of thrombosis on a structural level.A person new to this technique should avoid over-handling the tissue in the surgical area. This can lead to bleeding and can ...