Ferric Chloride-Induced Arterial Thrombosis in a Murine Model: Measurement of Occlusion and Sample Collection for Electron Microscopy

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 insult, 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 these players lead to either uncontrolled thrombosis or 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 is initiated and progresses in vivo. This model is based on endothelial damage/denudation and subsequent clot formation at the injury site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of inflicted injury. 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. In this article, we explain how to initiate and monitor FeCl₃-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

Introduction:

Thrombosis is a formation of a blood clot that partially or completely blocks the blood vessel impeding 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 1 in 4 deaths worldwide {Lozano, 2012 #18;Raskob, 2014 #19;Walton, 1979 #60}.

Although the thrombosis is manifested as a malfunction of vascular system, it could be a result of 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 systems including endothelial cells, blood cells, platelets and coagulation proteins. Upon vascular injury, platelets interact with adhesive proteins in subendothelial matrix and release their granular contents that recruit more platelets at the site. Concurrently, coagulation cascade gets activated that leads to fibrin formation and ultimately a clot is formed that contains platelets, and a few blood cells trapped within fibrin mesh. Although multitudes of antiplatelet and anticoagulant drugs are available to treat thrombosis, spurious bleeding still remains a major concern with these therapies requiring to fine tune the dosages and combinations of these drugs and urgent need to discover new ones.

Thrombosis is studied using multiple ways to inflict a vascular injury that includes 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 the extent of injury. Among all these types, FeCl₃ induced vascular injury is the most widely used method that has been employed in mouse, rats, rabbits and guinea pigs animal models.^3-6. This model is relatively simple, easy to use and if major parameters are established, is sensitive and reproducible in various vascular systems including artery (carotid and femoral), veins (jugular), arterioles (cremaster and mesenteric). (Xi at al., Thromb Haemost, 2014,)

In this manuscript, we describe protocol 1 to conduct basic FeCl₃- mediated vascular injury with minimal setting. In protocol 2 we describe the method to collect and fix the vascular injury sample for electron microscopy.

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

Materials:

Materials are listed in Figure 1.

Figure 1.

Surgical items needed to perform FeCl₃-induced carotid artery thrombosis in mice. (A) 1. LEICA S8AP0 microscope and stand. 2. Small animal heated pad. (B) 1. Gauze sponges. 2. 26G 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. Hair remover 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) 1. Transonic flow probe with flexible region (red arrow) and a notch that holds a vessel (black arrow).

Animals:

C57BL/6J mice 8-10 weeks old

Microscope and illuminator:

LEICA S8AP0 Microscope (LEICA) (no longer available) and LEICA S8AP0 Microscope stand (LEICA Cat no# 10447255)

Steromaster Illuminator (Fisher Scientific Cat no # 12-562-21)

Softwares:

WinDAQ^™/100 Software for Windows (DATAQ Instruments version 3.38)

ZEISS AxioCam Icc 1 (ZEISS Cat no# 57615)

Probe and flow system:

Doppler flowProbe (Transonic Systems Cat no# MA0.5PSB)

Research flowmeter (Transonic Systems Cat no# T402B01481)

Compact scale (Ward’s Science Cat no# 470314-390)

1 ml Syringe (BD& company, Cat no# 309659)

26G x 3/8 Needle (BD& company, Cat no# 305110)

2,2,2 Tribromoethanol (Sigma Aldrich Cat no # 48402)

2-methyl-2-butanol (Sigma Aldrich Cat no #240486)

Fixative:

Paraformaldehyde 16% solution (Electron microscopy Services Cat no # 15710)

Glutaraldehyde 10% solution (Electron microscopy Services Cat no # 16365)

L-Aspartic acid (Sigma Fisher Cat no# A93100)

Uranyl Acetate (Electron microscopy Services Cat no # 22400)

Sodium Cacodylate Buffer 0.2 M pH 7.4(Electron microscopy Services Cat no # 11623)

Thiocarbohydrazide (TCH) (SIGMA-ALDRICH at no # 88535)

Potassium Ferricyanide (SIGMA-ALDRICH at no # P-8131)

Osmium tetroxide 4% aqueous solution (Electron microscopy Services Cat no # 19150)

Lead nitrate (Fisher Scientific Cat no # L-62)

Ethyl Alcohol, anhydrous 200 proof (Electron microscopy Services Cat no # 15055)

Propylene Oxide, ACS reagent (Electron microscopy Services Cat no # 20401)

Araldite GY 502 (Electron microscopy Services Cat no # 10900)

EMBED 812 resin (Electron microscopy Services Cat no # 14900)

Dodenyl Succinic Anhydride/ DDSA (Electron microscopy Services Cat no # 13700)

DMP-30 activator (Electron microscopy Services Cat no # 13600)

Anesthesia:

CAUTION- Both reagents used to make the anesthetic solution- tribromoethanol and 2-methyl-2-butanol are toxic and irritant. Surgical gloves and mask should be used while handling these chemicals.

Make anesthetic solution by adding 2.5 gms of 2,2,2 Tribromoethanol (info) to 5 ml of 2-methyl-2-butanol (amylene hydrate/tertiary amyl alcohol). Then add this mixture to 200 ml of distilled water. Let it dissolve completely. Then filter the solution with 0.5 um filter and store in aliquots in dark containers at 4 °C. This solution should be used within two weeks. Tribromoethanol is light and heat-sensitive. Its degradation produces hepatotoxic and nephrotoxic byproducts.

Small Animal Heated Pad (K&H Manufacturing Inc. Model: HM10)

Scotch Magic Invisible Tape, 3/4" x 1000", Clear (Scotch, Cat no# 305289)

25 Yard Black Braided Silk Suture (5-0) (DEKNATEL Cat no# 136082-1204)

190 Proof Ethanol (KOPTEC Cat no# V1101) (used to make 70% ethanol)

Gauze Sponges 2” x 2” – 12 Ply (Dukal Corporation, Cat no# 2128)

7.5 mL Transfer Pipet, Graduated to 3 mL (Globe Scientific Inc., Cat no# 135010)

Filter paper (VWR, Cat no# 28310-106)

Sodium chloride (Fisher Scientific, Cat no# BP358-212)

Light-Duty Tissue Wipers (VWR, Cat no# 82003-822)

Iron (III) Chloride (anhydrous powder, molecular weight 162.2 g/mol) (SIGMA-ALDRICH Cat no# 157740)

Finger Loop Ear Punches (Fine Science Tools Cat no# 24212-01)

White Antistatic Hexagonal Weigh Boats, Medium, 64 X 15 X 19 mm (Fisher Scientific Cat no# S38975)

Sterile Cotton Tipped Applicators (Puritan Medical Products Cat no# 25-806 1WC)

Veet Gel Cream Hair Remover (Reckitt Benckiser Cat no# 3116875)

Alcohol Prep Pads (70% Isopropyl Alcohol) (Medline Cat no# MDS090735)

Dissecting Scissors, 12.5 cm long (World Precision Instrument Cat no# 15922-G)

Surgical Dumont #7 Forceps (Fine Science Tools Cat no# 11271-30)

Eye Dressing Forceps, 4" Full Curved, Standard, 0.8mm Wide Tips (Integra Miltex Cat no# 18-784)

Micro Dissecting Forceps; 1x2 Teeth, Full Curve; 0.8 mm Tip Width; 4" Length (Roboz Surgical Instrument Company Cat no# RS-5157)

Fine Scissors - Sharp-Blunt (Fine Science Tools Cat no# 14028-10)

Rainin Classic Pipette PR-10 (Rainin Cat no# 17008649)

Universal Low Retention Pipet Tip Reloads (0.1-10 μL) (VWR Cat no# 76323-394)

Doggy Poo Bags (Crown Products Cat no# PP-RB-200)

Knife Handle Miltex^® Extra Fine Stainless-Steel Size 3(Integra Lifesciences Cat no# 157510)

Integra^® Miltex^® Carbon Steel Surgical Blade #10 (Integra^® Miltex^® Cat no# 4110)

Cell Culture Dish 35mm X 10mm (Corning Inc, Cat no# 430165)

Fixative solution:

Caution- both paraformaldehyde and glutaraldehyde are highly toxic and irritants. While handling them, gloves, eye shield and masks should be used to protect from exposure.

Make 3% paraformaldehyde and 0.1% glutaraldehyde solution in filtered 1X PBS.

Procedure:

Protocol 1: FeCl₃- induced carotid artery injury

A. Anesthetize the mouse

1. Weigh the mouse.

2. Anaesthetize the mouse by injecting 0.2 g/Kg tribromoethanol solution intraperitoneally. Make sure that the anesthetic solution is at room temperature before injecting it. This dose should sedate the mouse for about an hour. Check the toe reflex by pinching the toe after 5 minutes of injecting to make sure mouse is sedated.

   If mouse is not sedated, it will pull away the toe. If that happens, wait for 5 more minutes for sedatives to settle in and check again. If the mouse is still not completely sedated, administer 1/4^th of the initial dose and wait for 5 minutes. Throughout the procedure, monitor the sedation of the mouse.

B. Immobilize the mouse

1. Lay mouse on the heating pad (37 °C) in supine position and use adhesive tape to immobilize the extremities.

2. Use a surgical thread to gently pull upper front teeth of the mouse so that the cervical/neck region is extended. This is important for the better visualization of the surgical area and the stability of the Doppler probe.

C. Incision

1. Spray the surgical area with 70% ethanol or wipe it with alcohol wipe.

2. Then using a cotton swab, apply a small amount of hair removing cream on the surgical area. Wait for 1-2 minutes and then use a wet paper towel or tissue to remove the cream. The hair will be removed from the cervical region. This step is important for the clean surgical area.

3. Using scissors and forceps (Fig. 1B11 and 13), take a midline incision from the mandible to the suprasternal notch. Retract the skin to get better visualization of the area (Fig. 2A).

Figure 2:

Steps in FeCl₃- induced carotid artery injury to measure vessel occlusion.

Using the scissor and surgical forceps take a midline section and gently pull away the tissue (A) to expose carotid artery beneath (B). The black arrow shows the direction of blood flow (B). Clean the surrounding tissue encircling carotid artery (C) and place a plastic paper (yellow plastic shown by a black arrow) and the probe (D). Perform FeCl₃ injury by placing FeCl₃ soaked filter paper (shown by red arrow) (E) on the artery (M). Remove the paper and monitor the injury. At the end, the injury is visible as yellow-white streak indicated by blue arrowhead (F).

D. Carotid artery Exposure

1. Using surgical forceps and micro dissecting forceps, blunt dissect the overlaying superficial fascia and remove it to expose left carotid artery (Fig. 1B12 and 13).

   Make sure not to use scissor at this stage to avoid injuring other vasculature in this area.

2. Remove extra tissue surrounding the carotid artery.

   Avoid too much dissection of the surrounding tissue since this area harbors Vagus nerve and vertebral artery. Separate the carotid artery from surrounding tissue by dissecting it with surgical and suture tying forceps. (Fig. 1B12 and 15, 2C) Make sure not to extend or pull the artery to avoid any mechanical injury to the artery or the surrounding vasculature.

3. Place the piece of plastic under the artery to mark the location of injury (Fig. 2D).

E. Placement of the Flow probe

1. The probe should be in saline solution for about 10 minutes before the surgery.

   When it is not in use, it should be in saline. Make sure to keep the probe moist and clean throughout the procedure.

2. Place the ultrasound Doppler transonic flow probe around the artery upstream of the plastic (Fig. 2D). Make sure that the vessel holder region of the probe is not too extended (Fig. 1C, red arrow).

   If needed, support the probe with gauze pads (Fig. 1B1) to achieve appropriate height of the probe to facilitate optimal reading position.

3. If the surgical area is dry by this point, add a few drops of room-temperature saline to keep the area moist. Monitor the flow probe reading. Optimal reading varies for each animal and could be anywhere between 0.6-1.2 ml/min. If it less or if it is not stable, change the flow probe position to get optimal reading.

   Make sure that the neck of the mouse is not too extended and surgical area is clean.

F. Record the flow

1. After placing the probe, monitor the blood flow for 2 min to make sure that the flow is steady. Then record the flow for 2 min (Fig. 3B) as a baseline. For detailed description of the flowmeter refer to ⁷.

2. To record blood flow, start WinDAQ software. Then click “file” option and then click on “record” option. This will be directed to an option of making a folder. Make a new folder and file. Then start recording. When you want to stop recording, click on “stop” option in file section.

Figure 3:

Blood flow readout using flowmeter.

The blood flow in carotid artery is measured using flowmeter (A). The flow is recorded using a record function in file tab (B). The baseline flow should be relatively constant before conducting the injury (B). After the injury the blood flow decreases uniformly (C).

G. Injury

1. Stop the recording of the flow and dry the area with a folded paper towel/ wipe.

   Make sure not to alter the probe position so that the readings stay consistent after the injury.

2. Dry the area thoroughly with wipe/paper towel.

   Even small volume of residual liquid may interfere with the composition of FeCl₃ used to inflict the vascular injury. This may lead to variable results and affect reproducibility. When the area is completely dry, probe will not be able to measure the flow.

3. In a plastic weighing boat put a circular filter paper (1mm diameter, cut with 1 mm diameter ear punch). Add 1 ul of 6% FeCl₃ solution on the top of the filter paper.

   Make sure to add FeCl₃ just before you use the filter paper. Soaking the paper early may lead to evaporation of FeCl₃ and changing the severity of the injury.

4. Using fine forceps, pick up the filter paper and put it on the artery downstream of the probe, on the part of artery marked by the plastic (Fig. 2D and E). This plastic acts as a marker and a spacer. It marks the injury area and also prevents accidental dilution of the FeCl₃ on filter paper by avoiding the contact with the surrounding moist area.

   Make sure that the filter paper is straight, not pinched or otherwise folded in any way. Once placed on the artery, do not change the position of the filter paper in any way. Doing so will change the extent of the injury.

5. After placing the filter paper, start the timer and record the blood flow. After 3 minutes of placing the filter paper, Remove the paper and add the saline at the site of injury to remove the FeCl₃ and stop the injury process. It also makes the area moist. At this stage, the blood flow should return to the baseline level as recorded pre-injury.

6. Monitor and record the flow till it reduces to 10% of the original recording or reaches to zero. There should be steady decrease once thrombus starts to form at the injury site (Fig. 3C). Note any significant fluctuations in the flow during this time. To study the stability of the thrombus, record the rapid increase in blood flow after significant and consistent decrease. These events could be classified as embolization due to unstable thrombus formation (Figure 4D). After the consistent decrease in flow, you may be able to see the injury on the artery at the termination of the experiment (blue arrow/dotted oval, Figure 2F).

7. The vessel occlusion time is defined as the complete cessation of blood flow for at least 1 min. Record the time at which occlusive thrombus formed as shown by the zero reading or significant decrease in the flow.

8. The experiment is terminated at 30 min. If the thrombus does not form by 30 minutes of monitoring, then stop the recording, remove the probe and clean it with 70% ethanol and a brush to remove any residual tissue/hair or debris. Dry it completely before placing it in the storage box.

9. Euthanize the mouse by cervical dislocation. Place the carcass in the carcass disposal bag labelled with the lab's name and the date.

Figure 4:

Data representation of FeCl₃-induced vascular injury.

Various methods to present data from FeCl₃-induced vascular injury are shown. Kaplan-Meier survival curves show the time to occlusion for each mouse. Log-rank test is performed for statistical analysis (A). The blood flow at the end of the experiment could also be represented in a bar graph (B) or line graph (C). Example of potential embolization event by sudden increase in blood flow after sustained gradual decrease in flow.

Protocol 2: Collection and Preparation of samples for EM studies post FeCl₃- induced injury

When collecting a vessel sample of FeCl₃-induced injury for EM analysis, following changes need to be made while performing the surgery-

1. After exposing the carotid artery, mark the site of injury by loosely encircling the region between two surgical threads (Fig. 5A and B).

2. Place the probe under the artery distal from the upper thread (Fig. 5C).

3. Insert the plastic piece under the artery between the two threads so as to mark the site for FeCl₃ injury (Fig. 5C).

4. Take flow measurements before performing injury to note the basal flow. These measurements will be used to decide at which stage you want to collect the sample.

   Make sure fixative (3% paraformaldehyde +0.1% glutaraldehyde) is ready and is at room temperature (RT).

5. Perform the injury by placing FeCl₃ soaked filter paper on the artery (we used 8% FeCl₃) for 3 min (this time could vary as well). After 3 min, remove the filter paper and add saline at the injury site to remove any excess residual FeCl₃. This step is necessary to prevent variable extent of injury and to facilitate flow counting by the probe (Fig. 5D).

6. Monitor the flow reading. When the flow drops 50% of the initial value, remove the probe. Quickly dry the area and add fixative in the area to externally fix the injury area.

7. Promptly hold the artery near the injury area with the forceps and 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 few drops of the fixative.

8. Using scalpel and blade, clean the extra tissue around the artery. Be mindful that you record the orientation of the blood flow. To mark that, cut one end horizontal and the other tapering/oblique (Fig. 5E).

9. Keep the sample in fixative (3% PFA and 0.1% glutaraldehyde) for one hour at room temperature. Then store the sample in 1% PFA at 4 °C until sending it on ice to the sample processing lab for EM analysis.

Figure 5:

Sample collection of FeCl₃-induced injury clots for EM studies.

After exposing an artery (A), mark the injury site with loosely tying the threads (B) 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 text and clean the sample and cut the sample straight on one side and oblique on the other side to show the direction of blood flow (black arrow shows the direction of blood flow and the red outline indicates the injured region) (E).

Cutting the artery will result in immediate extensive bleeding so make sure to hold the artery before cutting it the first time. In this scenario, the mouse will be sacrificed by exsanguination.

The caveats of this method of sample collection are:

A. Initial reading may not be optimal or may fluctuate after the injury. This may affect the extent of injury that you want to arrest for EM analysis. To address this problem, make sure that you record baseline reading for a few minutes after trying various positions of placing the probe to decide which position is optimal.

B. The time to arrest the injury by adding external fixative and actual cutting the arterial section for analysis may add variability of thrombus extent. It is immensely important to be quick in sample collection after fixing externally.

• 10.After initial 1hr fixation in 3% paraformaldehyde (PFA)/01% glutaraldehyde (GA) rinse the samples with 0.1 M cacodylate buffer. (for how long? RT?)
• 11.Fix the samples for 1 hr. on ice in 0.1 M Cacodylate buffer + 2.5% glutaraldehyde + 2mM Calcium chloride.
• 12.Discard the fixative after an hour and wash the samples with cold 0.1 M sodium cacodylate buffer containing 2mM CaCl₂ (5x 3min) (Fig. 6A).
• 13.Then fix the samples with Osmium solution (3% Potassium ferrocyanide + 0.3 M cacodylate buffer + 4 mM CaCl₂ mixed with equal volume of 4% aqueous OsO₄) for 1 hr. on ice.
• 14.While samples are fixing, make fresh 1% TCH (Trichloroacetaldehyde hydrate) solution in ddH₂0. Incubate the solution at 6 °C for 1hr while gently mixing.
• 15.After fixing, wash the samples with ddH₂O at RT for 5x 3 min.
• 16.Then incubate samples in filtered 1% TCH solution (Made in ddH₂0) for 20 min at RT.
• 17.Wash samples with ddH₂O at RT (3 min x 5).
• 18.Fix samples in 2% Osmium tetroxide in ddH₂O for 30 min at RT.
• 19.Wash samples with ddH₂O at RT each for 3 min x 5.
• 20.Place samples in 1% uranyl acetate (aqueous) and incubate overnight at 4 °C.
• 21.Wash samples with ddH₂O at RT each for 3 min x 5 and process them with en bloc Walton’s lead aspartate staining.
• 22.En bloc Walton’s lead aspartate staining {Walton, 1979 #60}:

  Make 30 mM L-aspartic acid (Sigma Fisher Cat no # A93100) solution in ddH₂O.

  Note: the aspartic acid will dissolve more quickly if the pH raised to 3.8. This stock solution is stable for 1-2 months if refrigerated.

  Make 20 mM lead nitrate solution in 10 ml of aspartic acid stock and pH is adjusted to 5.5 with 1N KOH.

  Samples are placed in lead aspartate solution at 60 °C for 30 min following five washed with ddH₂O at RT each for 3 min.

• 23.Dehydrate the specimens with a graded ethanol series.

  Chemical dehydration by Valdivia protocol: (reference for Valdivia protocol?)

  Wash the sample in each of the following solution to progressively dehydrate the sample (Fig. 6B).

  • 25% ethyl alcohol for 3 min
  • 50% ethyl alcohol for 3 min
  • 75% ethyl alcohol for 3 min
  • 95% ethyl alcohol for 3 min
  • 100% ethyl alcohol for 10 min and repeat the step twice (total 3 washes).
  • 100% Propylene Oxide for 10 min and repeat the step twice (total 3 washes).
• 24.Incubate in 50%/50% PO/resin with DMP 30 activator overnight at RT and embed in 100% Araldite 502/embed 812/DDSA with DMP30 activator for 48 hours.

Figure 6:

Post fixation processing of samples for Electron microscopy.

The samples are washed in microcentrifuge tubes between processing steps (A) and serially dehydrated with 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 in slices for imaging.

Data Presentation

The data are generally presented as time to occlusion/time it takes to form a fully occlusive thrombus that impedes the blood flow. These data could be plotted as survival curve (Fig. 4A), dot plot with bars showing the terminal blood flow at the time of either cessation of the blood flow or at the termination of an experiment (Fig. 4B) or as a line graph (Fig. 4C). The thrombus stability could 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 to zero upon complete occlusion of the vessel. In some cases, the blood flow suddenly increases following gradual decrease for a few minutes. This could be interpreted as partial shedding of the growing thrombus and could be considered as embolization event (Fig. 4D). Occlusive thrombus morphology (Fig. 7A) could be studied using this method.

Figure 7:

EM image of fully occlusive thrombus post FeCl₃-mediated carotid injury.

A complete transverse section of occluded artery showing vascular wall (A) and insets showing structure at higher magnification, scale bars- 20 μm (B, C & D),

Discussion:

The topical application of FeCl₃ to vasculature to induce thrombosis is a widely used technique and it has been instrumental in establishing roles of various platelet receptors, ligands signaling pathways and their inhibitors ^{8 9 10 11}. The mechanism through which FeCl₃ causes thrombosis is multifaceted. Previously, endothelial denudation was considered a cause for thrombosis, however in recent years multiple reports suggested the role of RBCs and plasma proteins in this process ^{12 13 14}{Shim, 2021 #54}.

Though simple and sensitive, this technique could 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, sex and type of anesthesia used. These differences may be accountable for the relatively wide range of thrombosis time in C57Bl6/J reported in literature (Supplemental Table 1)^15-19{Shim, 2021 #54}.

In this manuscript, we provide detailed procedure to minimize data variations and increase reproducibility. We have provided a table to troubleshoot a few problems that may arise during this surgery (Table 1). Additionally, we also propose a method to collect samples at the end of injury to study morphology of occlusive thrombus (Fig. 7). Another important advantage of this technique is that you will be able to use baseline readings to decide at what stage you want to collect thrombus sample. Be mindful that these are approximate timings and do not indicate the exact extent of injury/thrombosis.

Table 1:

Potential technical challenges in FeCl₃-induced thrombosis model and solutions

Problem	Cause	Solution
Sedation is not complete. Toe pinch reflex present after the IP injection of avertin	Anesthesia is insufficient.	Make sure to weigh the mouse and inject appropriate volume. Wait for 5 more minutes. If sedation is still not complete, administer 1/4th of initial volume.
Cannot find carotid artery	Excessive handling of neighboring tissue	Use trachea as a marker and then look for the artery on the lateral side that is round and smooth. The area is rich with small arteries but the size difference should help identify carotid artery.
No flow reading	The surgical area is not clean.	Remove the encasing membranes from the carotid artery and then use fine forceps to separate the carotid artery from the surrounding tissue.
No flow reading	The surgical area is dry and there is not much fluid where the probe is placed.	Add a few drops of room temperature saline in the surgical area so that the probe is merged in the saline.
No flow reading/inconsistent flow	Doppler probe is not clean.	Soak the probe in the saline for 30 minutes and remove any tissue stuck in the probe completely. Make sure to clean the probe thoroughly with saline after the surgeries.
The flow is not optimal	The cervical region is too extended and the artery is stretched.	Make sure that the cervical region is not too extended and the artery is not stretched by keeping the upper extremities in physiological position.
The flow fluctuates	The probe is unstable.	Stabilize the probe by using gauze pads to elevate the probe at optimal height or to support the probe to avoid its tilting on one side.
The flow fluctuates	Sedation is not complete and the mouse hyperventilates.	Inject the mouse with 1/4th of initial dose of Avertin and support the probe so that the thoracic movement will not move the probe.
Data variation	FeCl3 concentration is not right.	Make the FeCl3 solution fresh for the surgery. Make sure all the calculations are right.
Data variation	FeCl3 is diluted due to the residual liquid in the surgical area.	Use a wipe or folded paper towel to remove all the liquid in the surgical area so that the area is completely dry.
Data variation	FeCl3 concentration is not right.	The size of the filter paper should be uniform throughout the project. Different sizes will change the intensity of the injury leading to variability in data.
Data variation	Filter paper is not optimally placed.	The filter paper should not be curved and it should be completely soaked with FeCl3 solution. Put filter paper on the artery immediately after soaking it to avoid evaporation.
Data variation	Filter paper is not optimally placed.	Make sure the filter paper is in complete contact with the artery. Do not change the position of filter paper once placed on the artery. Changing the position may create a streak of injury and the injury will not be consistent leading to variability in thrombosis.
Data variation	Improper removal of FeCl3 post-injury	Make sure to wash the surgical area properly to remove excess FeCl3 to avoid variable thrombosis.

This is a minimal required setting 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 platelet releasate such as PF4 and P-Selectin and fibrin to get a better visualization of the in vivo thrombus {Li, 2013 #53} {Li, 2016 #31}. This technique could also help to monitor embolization events that indicate thrombus instability (Fig. 4D).