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 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.
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
2. Collection and preparation of samples for serial block face scanning electron microscopy (SBF-SEM) studies post FeCl3-induced Injury
NOTE: The EM protocol presented is appropriate for sample preparation for SBF-SEM. This imaging technique offers an unprecedented ability to study the three-dimensional structure of platelets in a clot. With this technique, the sample is visualized as a series of sequential block SEM images, generated as one progresses through the sample. Key points for this preparation are: 1) the sample must be stained with heavy metals pre-embedding; and 2) the plastic embedded sample must be trimmed appropriately for mounting within the SEM (see Figure 6 for a visual of trimmed samples). Post-embedding staining is not possible within the SEM. When collecting a sample from a FeCl3-induced vessel injury for EM analysis, the following changes need to be made while performing the surgery. The modifications in the FeCl3 surgery protocol presented are also applicable to any form of electron microscopy. Anesthesia conditions and the steps for making an incision and exposing the carotid artery are the same as in Protocol 1.
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 technique. In most cases, upon FeCl3 injury, the thrombus forms gradually, and as it grows, the blood flow progressively decreases, reaching zero upon complete occlusion of the vessel. In some cases, the blood flow suddenly increases following a gradual decrease for a few minutes. This is interpreted as partial shedding of the growing thrombus, and can be considered as an embolization event (Figure 4D). Occlusive thrombus morphology (Figure 7A) can be studied using this method as well.
Table 1: Potential technical challenges in the FeCl3-induced thrombosis model and solutions. Please click here to download this Table.
Figure 1: Surgical instruments needed to perform FeCl3-induced carotid artery thrombosis in mice. (A) 1: LEICA S8AP0 microscope and stand. 2: Small animal heated pad covered in aluminum foil. (B) 1: Gauze sponges. 2: 26 G x 3/8 needle. 3: 1 ml syringe. 4: Sterile cotton-tipped applicators. 5: Black braided silk suture. 6: Surgical blade. 7: Stainless steel knife handle. 8: Commercial hair removal cream. 9: Ear punch. 10: Dissecting scissors. 11: Fine scissors. 12: Surgical forceps. 13: Micro dissecting forceps. 14: Eye dressing forceps. 15: Suture tying forceps. (C) Transonic flow probe with the flexible region (red arrow) and a notch that holds a vessel (black arrow). Please click here to view a larger version of this figure.
Figure 2: Surgical steps to prepare the carotid artery for injury and measure vessel occlusion. Using scissors and surgical forceps, make a midline section and gently pull away the tissue (A) to expose the carotid artery beneath it (B). The black arrow shows the direction of blood flow (B). Clean the surrounding tissue encircling the carotid artery (C) and place a piece of plastic paper (yellow plastic shown by a black arrow) and the probe (D). Injure the vessel by placing FeCl3-soaked filter paper (shown by a red arrow) on the artery (E). Remove the paper and monitor the injury. At the end, the injury is visible as a yellow-white streak, indicated by the blue arrowhead (F). Please click here to view a larger version of this figure.
Figure 3: Blood flow readout using the flowmeter. The blood flow in the carotid artery is measured using a flowmeter (A). The flow is recorded using a record function in the file tab (B). The baseline flow should be relatively constant before conducting the injury (about 0.8 mL/min, shown by a black arrow) (B). After the injury, the blood flow decreases uniformly (about a 50% decrease from the starting value, about 0.4 mL/min), shown by a black arrow (C). Please click here to view a larger version of this figure.
Figure 4: Representative data presentations from the FeCl3-induced carotid injury model. Various methods available to present data from FeCl3-induced vascular injury are shown. Kaplan-Meier survival curves show the time to occlusion for each mouse. A log-rank test is performed for statistical analysis. *** p ≤ 0.001 (A). The blood flow at the end of the experiment could also be represented in a bar graph (B). For the data represented in B, an unpaired t-test was performed *** p ≤ 0.001. The error bar represents mean ± SD. The Doppler flow data collected post-injury from a single animal could be presented in a line graph (C). Example of a potential embolization event by the sudden increase in blood flow after a sustained gradual decrease in flow in a single mouse at various time points (D). Please click here to view a larger version of this figure.
Figure 5: Collection of FeCl3-induced injury clots for electron microscopy studies. After exposing an artery (A), mark the injury site by loosely tying the threads (B), and place the plastic paper under the artery and the probe downstream of it (C). Perform the injury and monitor the damage (D). Collect the tissue as explained in the text, and clean and cut the sample straight on one side and oblique on the other side to show the direction of blood flow (the black arrow shows the direction of blood flow and the red outline indicates the injured region) (E). Please click here to view a larger version of this figure.
Figure 6: Post-fixation processing and mounting of samples for electron microscopy. The samples are washed in microcentrifuge tubes between processing steps (A) and serially dehydrated with an increased concentration of ethanol (B). After processing, they are embedded in resin (C), and the blocks are marked with the direction of the blood flow (D) and then cut into slices for imaging. Please click here to view a larger version of this figure.
Figure 7: Representative electron micrographs of proximal and distal regions of a fully occlusive thrombus post FeCl3-mediated carotid injury. (A) A complete transverse section of an occluded artery, proximal to the injury site, with insets below, showing structures at higher magnification (a: 2x, and a': 4x). The injured area is indicated with an arrow in A. Scale bar: 100 µm. (B) A complete transverse section of an occluded artery, distal to the injury site, with insets below, showing structures at higher magnification (b: 2x, and b': 4x). Please click here to view a larger version of this figure.
Supplemental Table 1: A brief survey of variations in animal models, FeCl3 injury site, FeCl3 concentration, injury method, and thrombosis times. Please click here to download this File.
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 thrombosis, however in recent years, multiple reports have suggested the role of red blood cells and plasma proteins in this process24,25,26,27.
The most critical steps in this protocol are: the placement of the Doppler probe to get an optimal flow, the placement of the filter paper, and the prompt termination of the injury to retrieve and immediately fix the sample. The consequences of mishaps in these steps and how to address them are described in Table 1. Though simple and sensitive, this technique can be challenging to employ, depending on the animal model, animal background, site of vascular injury, concentration of FeCl3, FeCl3 application method, duration of application, animal age and sex, and type of anesthesia used. These differences may account for the relatively wide range of thrombosis times in C57BL/6J reported in the literature (Supplemental Table 1)27,28,29,30,31,32. This protocol proposes to use 8-10-week-old mice for the experiment, as the vasculature size is easier to visualize and surgerize. Some groups have used mice as young as 6 weeks old33. It is imperative to age-match the mice for consistent and reproducible comparison among various animal groups. The anesthesia described, tribromoethanol, is preferred because it is readily available, is easier to administer, it maintains an anesthetic plane for the required duration, and the dosage required is reproducible. Many other anesthetic options are available, including isoflurane and various combinations of tribromoethanol with tertiary amyl alcohol with xylazine and/or acepromazine. It is imperative to use an optimum dose to achieve sedation without negatively affecting the heart rate or blood pressure of the animal. Using the same method of anesthesia throughout the experiment prevents potential compounding effects on the thrombosis time.
This manuscript presents a detailed procedure to minimize data variations and increase reproducibility. A table is provided to troubleshoot a few problems that may arise during this surgery (Table 1). Additionally, this manuscript proposes a method to collect samples at the end of injury to study the structure and morphology of an occlusive thrombus (Figure 7). The resolution of EM offers a better visualization than was previously possible25 of platelets in a growing thombus, and how they interact with other platelets and the endothelium both proximal and distal of the vascular injury. It can also be used to study various activation stages of platelets at the injury site.
Another important advantage of this technique is that the operator can use baseline readings to decide at what stage the thrombus sample is collected. It should be noted that these are approximate timings and do not indicate the exact extent of injury/thrombosis. Basal readings are dependent on the optimal placing of the probe, and if not placed correctly, the readings may record only partial flow. This leads to faulty interpretation of the termination time and therefore morphology of the collected sample. Prompt termination of the injury process and immediate fixation of the samples are required to achieve consistent and reproducible results.
This protocol presents the minimal required settings for the evaluation of occlusive thrombosis. With advances in microscopy, several groups have used this technique to fluorescently label the platelets/endothelial cells and/or the platelet releasate, such as PF4 and P-Selectin and fibrin, to visualize specific aspects of in vivo thrombosis and to determine its kinetics34,35. As described, the method here can report on embolization events, which indicate thrombus instability (Figure 4D).
The authors have nothing to disclose.
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.
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; 1×2 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 |