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

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 worldwide^1,2,3. 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 dogs^8,9,10,11,12. 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 agents^14,15. 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.

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

1. Mouse anesthesia induction
   1. Weigh the mouse.
   2. Anesthetize the mouse by injecting 0.2 g/kg tribromoethanol solution intraperitoneally (i.p.). Make sure that the anesthetic solution is at room temperature (RT) before injecting it. This dose is enough to sedate the mouse for about 1 h.
   3. Check the toe reflex by pinching the toe 5 min after injection to make sure the mouse is sedated. If the mouse is not sedated, it will pull its toe away. If that happens, wait 5 min and check again.
   4. If the mouse is still not completely sedated, administer 1/4 of the initial dose and wait for 5 min.
   5. Throughout the procedure, periodically monitor the anesthetic plane of the mouse via a toe-pinch. Additionally, respiration rate and body temperature could also be monitored to maintain optimum anesthesia.
2. Immobilize the mouse
   1. Lay the mouse on the heating pad (37 °C) in a supine position and use adhesive tape to immobilize the extremities.
   2. Use a surgical thread (0.1 mm) to gently pull the upper front teeth of the mouse to extend the cervical/neck region. This is important for better visualization of the surgical area and stability of the Doppler probe. The thread size is not important, as it is only used to pull the front teeth of the animal to extend the neck.
3. Incision
   1. Spray the surgical area with 70% ethanol or wipe it with an alcohol wipe.
   2. Using a cotton swab, apply a small amount of hair-removal cream on the surgical area. Wait for 1-2 min and then use a wet paper towel or tissue to remove the cream.
      NOTE: This step is important for a clean surgical area.
   3. Using scissors and forceps (Figure 1B11,1B13), make a midline incision from the mandible to the suprasternal notch. Retract the skin to obtain a better visualization of the area (Figure 2A).
4. Carotid artery exposure
   1. Using surgical forceps and micro dissecting forceps, blunt dissect the overlaying superficial fascia and remove it to expose the left carotid artery (Figure 1B12,1B13). Make sure not to use scissors at this stage to avoid injuring other vasculature in this area.
   2. Remove extra tissue surrounding the carotid artery. Avoid excessive dissection of the surrounding tissue, since this area harbors the vagus nerve and vertebral artery.
   3. Separate the carotid artery from surrounding tissue by dissecting it with surgical and suture-tying forceps (Figure 1B12,1B15,2C). Make sure not to extend or pull the artery to avoid any mechanical injury to the artery or the surrounding vasculature.
   4. Place a piece of plastic under the artery to mark the location of the injury (Figure 2D). Use a colored transparency sheet to make it easier to see in the surgical field.
5. Placement of the flow probe
   1. Place the Doppler transonic flow probe in saline solution (0.9% NaCl in dH₂O) for about 10 min before the surgery. Insert the other end of the probe into an attachment in the flowmeter to facilitate the reading of the flow. Attach the flowmeter to a computer.
      NOTE: In between surgeries, the Doppler transonic flow probe 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 (Figure 2D). Make sure that the vessel holder region of the probe is not too extended (Figure 1C, red arrow).
      NOTE: If needed, support the probe with gauze pads (Figure 1B1) to achieve an appropriate height of the probe, facilitating the optimal reading position.
   3. If the surgical area is dry by this point, add a few drops of RT saline to keep the area moist. Monitor the flow probe reading. The optimal reading varies for each animal and could be anywhere between 0.6-1.2 mL/min. If it is less or not stable, change the flow probe position to get an optimal reading.
      NOTE: Make sure that the neck of the mouse is not too extended, and that the surgical area is clean.
6. Record the flow baseline
   1. After placing the probe, monitor the blood flow for 2 min to ensure that the flow is steady. Then, record the flow for 2 min (Figure 3B) as a baseline. For a detailed description of the flowmeter, refer to Subramaniam et al.¹⁷.
   2. To record the blood flow, start the WinDAQ software. Click on the File option and then click on Record. Make a new folder and file, and start recording. To stop recording, click on Stop in the file section.
      NOTE: WinDAQ software is freely available from the publisher: https://www.dataq.com/products/windaq/.
7. Injury
   1. Stop the recording of the flow. Make sure not to alter the probe position so that the readings stay consistent after the injury.
   2. Dry the area thoroughly with a wipe/paper towel.
      NOTE: Even a small volume of residual liquid may alter the concentration of FeCl₃ solution used to inflict vascular injury. This leads to variable results and affects reproducibility. When the area is completely dry, the probe is not able to measure the flow.
   3. In a plastic weighing boat, put a circular filter paper (1 mm diameter, cut with a 1 mm diameter ear punch). Add 1 µL of 6% FeCl₃ solution (made in dH₂O) onto the filter paper.
      NOTE: Make sure to add FeCl₃ solution just before using the filter paper. Soaking the paper too early may lead to the evaporation of FeCl₃ solution and change the severity of the injury.
   4. Using fine forceps, pick up the filter paper and put it on the artery upstream of the probe, on the part of the artery marked by the plastic (Figure 2D,E). This plastic acts as a marker and a spacer. It marks the injured area and prevents accidental dilution of the FeCl₃ by avoiding contact with the surrounding moist area.
      ​NOTE: 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; doing so changes the extent of the injury.
   5. After placing the filter paper, start the timer and record the blood flow. After 3 min, remove the paper and add 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 zero. There is a steady decrease once the thrombus starts to form at the injury site (Figure 3C). Note any significant fluctuations in the flow during this time.
   7. To study the stability of the thrombus, record the rapid increase in blood flow after each significant and consistent decrease. These events are classified as embolization due to unstable thrombosis (Figure 4D). After the consistent decrease in flow, the operator is able to see the injury on the artery at the termination of the experiment (Figure 2F, blue arrow/dotted oval).
   8. The vessel occlusion time is defined as the complete cessation of blood flow for at least 1 min. Record the time at which an occlusive thrombus is formed, as shown by the zero reading or significant decrease in flow.
   9. Terminate the experiment at 30 min. If the thrombus does not form within 30 min of monitoring, then record the flow, stop the recording, and remove the probe.
   10. Clean the probe with 70% ethanol and a brush to remove any residual tissue/hair or debris. Dry the probe completely before placing it in the storage box. Dry the probe completely before placing it in the storage box for long term storage.
   11. Euthanize the mouse by cervical dislocation. Place the carcass in the carcass disposal bag, labeled with the lab's name and the date.

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.

1. After exposing the carotid artery, mark the site of injury by loosely encircling the region between two surgical threads (size: 0.1 mm) (Figure 5A,B).
2. Place the probe under the artery, distal from the lower thread (Figure 5C).
3. Insert the plastic piece under the artery between the two threads to mark the site for FeCl₃ injury (Figure 5C).
4. Take flow measurements before performing injury to note the basal flow. These measurements are used to decide at which stage the sample will be collected.
   NOTE: Make sure the fixative (3% paraformaldehyde/PFA + 0.1% glutaraldehyde, both made in 1x PBS) is ready and at RT. Alternatively, fixation with 2.5% glutaraldehyde in a saline background could also be used.
   CAUTION: Both paraformaldehyde and glutaraldehyde are highly toxic and irritant. While handling them, gloves, an eye shield, and masks should be used to protect from exposure. All fixative solutions are toxic, and fixation steps should be carried out in a fume hood.
5. Perform the injury by placing FeCl₃-soaked filter paper on the artery (8% FeCl₃ is used) 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 the variable extent of the injury and to facilitate flow counting by the probe (Figure 5D).
6. 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 in the area to externally fix the injury area.
7. Promptly hold the artery near the injury area with forceps, and cut downstream of the lower thread and upstream of the upper thread. If necessary, ask another person to help cut one end. 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.
   NOTE: Cutting the artery causes immediate extensive bleeding, so make sure to hold the artery before cutting it the first time. In this scenario, the mouse is exsanguinated. The mouse is euthanized by exsanguination and cervical dislocation.
8. Using a scalpel, clean the extra tissue around the artery. Be mindful to record the orientation of the blood flow. To mark that, cut one end horizontally and the other tapering/oblique (Figure 5E).
9. Keep the sample in fixative (3% PFA and 0.1% glutaraldehyde) for 1 h at RT. Then, store the sample in 1% PFA at 4 °C until sending it on wet ice to the sample processing lab for EM analysis.
   NOTE: The samples should not be frozen. The challenges for this method of sample collection are:
   1. The initial reading may not be optimal or may fluctuate after the injury. This may affect the extent of injury that is chosen to arrest for EM analysis. To address this problem, make sure to record the baseline reading for a few minutes, after trying various positions of placing the probe to decide which position is optimal.
   2. The time to arrest the injury by adding external fixative and actual cutting of the arterial section for analysis may add variability to the extent of thrombosis. Be quick during sample collection after fixing externally.
10. Receive the samples for processing for EM analysis in 1% PFA on wet ice. Rinse the samples for 3 min, on ice, with 0.1 M cacodylate buffer to initiate further processing.
11. Fix the samples for 1 h on ice in 0.1 M cacodylate buffer + 2.5% glutaraldehyde + 2 mM CaCl₂.
12. Discard the fixative and wash the samples with cold 0.1 M sodium cacodylate buffer containing 2 mM CaCl₂ (5 x 3 min) (Figure 6A).
13. Fix the samples with osmium solution (3% potassium ferrocyanide [K₄C₆N₆Fe] + 0.3 M cacodylate buffer + 4 mM CaCl₂ mixed with an equal volume of 4% aqueous osmium tetroxide [OsO₄; in ddH₂O]) for 1 h on ice.
14. While the samples are fixing, make fresh 1% trichloroacetaldehyde hydrate (TCH) solution in ddH₂O. Incubate the solution at 60 °C for 1 h while gently mixing.
15. After fixing, wash the samples with ddH₂O at RT for 5 x 3 min.
16. Incubate the samples in filtered 1% TCH solution (made in ddH₂O) for 20 min at RT.
17. Wash the samples with ddH₂O at RT (5 x 3 min).
18. Fix the samples in 2% osmium tetroxide in ddH₂O for 30 min at RT.
19. Wash the samples with ddH₂O at RT for 5 x 3 min each.
20. Place the samples in 1% uranyl acetate (aqueous) and incubate overnight at 4 °C.
21. Wash the samples with ddH₂O at RT for 5 x 3 min each, and process them with en bloc Walton's lead aspartate staining.
22. En bloc Walton's lead aspartate staining³:
    1. Make 30 mM L-aspartic acid solution in ddH₂O.
       NOTE: The aspartic acid dissolves more quickly if the pH is raised to 3.8. This stock solution is stable for 1-2 months if refrigerated.
    2. Make 20 mM lead nitrate solution in 10 mL of aspartic acid stock, and adjust the pH to 5.5 with 1 N KOH.
    3. Place the samples in lead aspartate solution at 60 °C for 30 min, following five washes with ddH₂O at RT, each for 3 min.
23. Dehydrate the specimens with a graded ethanol series. Use the chemical dehydration by Valdivia protocol¹⁸.
    1. Wash the samples in each of the following solutions to progressively dehydrate the sample (Figure 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 of three washes).
24. Wash the samples in 100% propylene oxide (PO) for 10 min, and repeat the step twice (total of three washes).
25. 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 h.

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 inhibitors^20,21,22,23. The mechanism through which FeCl₃ causes thrombosis is multifaceted; previously, endothelial denudation was considered a cause of thro...

Materials

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