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>
	<li>Mouse anesthesia induction
	<ol>
		<li>Weigh the mouse.</li>
		<li>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.</li>
		<li>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.</li>
		<li>If the mouse is still not completely sedated, administer 1/4 of the initial dose and wait for 5 min.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Immobilize the mouse
	<ol>
		<li>Lay the mouse on the heating pad (37 &#176;C) in a supine position&#160;and use adhesive tape to immobilize the extremities.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Incision
	<ol>
		<li>Spray the surgical area with 70% ethanol or wipe it with an alcohol wipe.</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Carotid artery exposure
	<ol>
		<li>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.</li>
		<li>Remove extra tissue surrounding the carotid artery. Avoid excessive dissection of the surrounding tissue, since this area harbors the vagus nerve and vertebral artery.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Placement of the flow probe
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Record the flow baseline
	<ol>
		<li>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.</li>
		<li>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:&#160;https://www.dataq.com/products/windaq/.</li>
	</ol>
	</li>
	<li>Injury
	<ol>
		<li>Stop the recording of the flow. Make sure not to alter the probe position so that the readings stay consistent after the injury.</li>
		<li>Dry the area thoroughly with a wipe/paper towel. 
		NOTE: Even a small volume of residual liquid may alter the concentration of FeCl3&#160;solution used to inflict vascular injury. This leads to variable results and affects reproducibility. When the area is completely dry, the probe is not&#160;able to measure the flow.</li>
		<li>In a plastic weighing boat, put a circular filter paper (1 mm diameter, cut with a 1 mm diameter ear punch). Add 1 &#181;L of 6% FeCl3 solution (made in dH2O) onto the filter paper. 
		NOTE: Make sure to add FeCl3&#160;solution just before using the filter paper. Soaking the paper too early may lead to the evaporation of FeCl3&#160;solution and change the severity of the injury.</li>
		<li>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. 
		&#8203;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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.&#160;Dry the probe completely before placing it in the storage box for long term storage.</li>
		<li>Euthanize the mouse by cervical dislocation. Place the carcass in the carcass disposal bag, labeled with the lab&#39;s name and the date.</li>
	</ol>
	</li>
</ol>

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.
<ol>
	<li>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).</li>
	<li>Place the probe under the artery, distal from the lower thread (Figure 5C).</li>
	<li>Insert the plastic piece under the artery between the two threads to mark the site for FeCl3 injury (Figure 5C).</li>
	<li>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.&#160;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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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 &#176;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:
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Fix the samples for 1 h on ice in 0.1 M cacodylate buffer + 2.5% glutaraldehyde + 2 mM CaCl2.</li>
	<li>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).</li>
	<li>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.</li>
	<li>While the samples are fixing, make fresh 1% trichloroacetaldehyde hydrate (TCH) solution in ddH2O. Incubate the solution at 60 &#176;C&#160;for 1 h while gently mixing.</li>
	<li>After fixing, wash the samples with ddH2O at RT for 5 x 3 min.</li>
	<li>Incubate the samples in filtered 1% TCH solution (made in ddH2O) for 20 min at RT.</li>
	<li>Wash the samples with ddH2O at RT (5 x 3 min).</li>
	<li>Fix the samples in 2% osmium tetroxide in ddH2O for 30 min at RT.</li>
	<li>Wash the samples with ddH2O at RT for 5 x 3 min each.</li>
	<li>Place the samples in 1% uranyl acetate (aqueous) and incubate overnight at 4 &#176;C.</li>
	<li>Wash the samples with ddH2O at RT for 5 x 3 min each, and process them with en bloc Walton&#39;s lead aspartate staining.</li>
	<li>En bloc Walton&#39;s lead aspartate staining3:
	<ol>
		<li>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.</li>
		<li>Make 20 mM lead nitrate solution in 10 mL of aspartic acid stock, and adjust the pH to 5.5 with 1 N KOH.</li>
		<li>Place the samples in lead aspartate solution at 60 &#176;C for 30 min, following five washes with ddH2O at RT, each for 3 min.</li>
	</ol>
	</li>
	<li>Dehydrate the specimens with a graded ethanol series. Use the chemical dehydration by Valdivia protocol18.
	<ol>
		<li>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%&#160;ethyl alcohol for 10 min, and repeat the step twice (total of three washes).</li>
	</ol>
	</li>
	<li>Wash the samples in 100% propylene oxide (PO) for 10 min, and repeat the step twice (total of three washes).</li>
	<li>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.</li>
</ol>

<ol>
	<li>Lozano, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+mortality+from+235+causes+of+death+for+20+age+groups+in+1990+and+2010:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2010.">Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010.</a> Lancet. 380 (9859), 2095-2128 (2012).</li><li>Raskob, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis:+a+major+contributor+to+global+disease+burden.">Thrombosis: a major contributor to global disease burden.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 34 (11), 2363-2371 (2014).</li><li>Walton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lead+aspartate,+an+en+bloc+contrast+stain+particularly+useful+for+ultrastructural+enzymology.">Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology.</a> Journal of Histochemistry and Cytochemistry. 27 (10), 1337-1342 (1979).</li><li>Palta, S., Saroa, R., Palta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+coagulation+system.">Overview of the coagulation system.</a> Indian Journal of Anaesthesia. 58 (5), 515-523 (2014).</li><li>Joshi, S., Whiteheart, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuts+and+bolts+of+the+platelet+release+reaction.">The nuts and bolts of the platelet release reaction.</a> Platelets. 28 (2), 129-137 (2017).</li><li>Periayah, M. H., Halim, A. S., Mat Saad, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+action+of+platelets+and+crucial+blood+coagulation+pathways+in+hemostasis.">Mechanism action of platelets and crucial blood coagulation pathways in hemostasis.</a> International Journal of Hematology-Oncology and Stem Cell Research. 11 (4), 319-327 (2017).</li><li>Alexopoulos, D., Katogiannis, K., Sfantou, D., Lekakis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+antiplatelet+treatment+in+coronary+artery+disease+patients:+A+necessary+evil+or+an+overzealous+practice.">Combination antiplatelet treatment in coronary artery disease patients: A necessary evil or an overzealous practice.</a> Platelets. 29 (3), 228-237 (2018).</li><li>Kurz, K. D., Main, B. W., Sandusky, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+arterial+thrombosis+induced+by+ferric+chloride.">Rat model of arterial thrombosis induced by ferric chloride.</a> Thrombosis Research. 60 (4), 269-280 (1990).</li><li>Denis, C. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+of+in+vivo+thrombosis+studies+in+mice.">Towards standardization of in vivo thrombosis studies in mice.</a> Journal of Thrombosis and Haemostasis. 9 (8), 1641-1644 (2011).</li><li>Marsh Lyle, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+thrombin+inhibitor+efficacy+in+a+novel+rabbit+model+of+simultaneous+arterial+and+venous+thrombosis.">Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis.</a> Thrombosis and Haemostasis. 79 (3), 656-662 (1998).</li><li>Kato, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+arterial+thrombosis+by+a+protease-activated+receptor+1+antagonist,+FR171113,+in+the+guinea+pig.">Inhibition of arterial thrombosis by a protease-activated receptor 1 antagonist, FR171113, in the guinea pig.</a> European Journal of Pharmacology. 473 (2-3), 163-169 (2003).</li><li>Huttinger, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride-induced+canine+carotid+artery+thrombosis:+a+large+animal+model+of+vascular+injury.">Ferric chloride-induced canine carotid artery thrombosis: a large animal model of vascular injury.</a> Journal of Visualized Experiments. (139), e57981 (2018).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithrombotic+therapy+by+regulating+the+ROS-mediated+thrombosis+microenvironment+and+specific+nonpharmaceutical+thrombolysis+Using+Prussian+blue+nanodroplets.">Antithrombotic therapy by regulating the ROS-mediated thrombosis microenvironment and specific nonpharmaceutical thrombolysis Using Prussian blue nanodroplets.</a> Small. 18 (15), 2106252 (2022).</li><li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+membrane+cloaked+nanotubes+to+accelerate+thrombolysis+by+thrombus+clot-targeting+and+penetration.">Platelet membrane cloaked nanotubes to accelerate thrombolysis by thrombus clot-targeting and penetration.</a> Small. , 2205260 (2022).</li><li>Refaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+light-responsive+liposomes+for+protein+delivery:+Towards+bleeding-free+photothermally-assisted+thrombolysis.">Near-infrared light-responsive liposomes for protein delivery: Towards bleeding-free photothermally-assisted thrombolysis.</a> Journal of Controlled Release. 337, 212-223 (2021).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+nanoplatelets+to+target+delivery+hirudin+for+site-specific+photothermal/photodynamic+thrombolysis+and+preventing+venous+thrombus+formation.">Biomimetic nanoplatelets to target delivery hirudin for site-specific photothermal/photodynamic thrombolysis and preventing venous thrombus formation.</a> Small. 18 (51), 2203184 (2022).</li><li>Subramaniam, S., Kanse, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride-induced+arterial+thrombosis+in+mice.">Ferric chloride-induced arterial thrombosis in mice.</a> Current Protocols in Mouse Biology. 4 (4), 151-164 (2014).</li><li>Cocchiaro, J. L., Kumar, Y., Fischer, E. R., Hackstadt, T., Valdivia, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+lipid+droplets+are+translocated+into+the+lumen+of+the+Chlamydia+trachomatis+parasitophorous+vacuole.">Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole.</a> Proceedings of the National Academy of Sciences. 105 (27), 9379-9384 (2008).</li><li>Kaplan, E. L., Meier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonparametric-estimation+from+incomplete+observations.">Nonparametric-estimation from incomplete observations.</a> Journal of the American Statistical Association. 53 (282), 457-481 (1958).</li><li>Chauhan, A. K., Kisucka, J., Lamb, C. B., Bergmeier, W., Wagner, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Willebrand+factor+and+factor+VIII+are+independently+required+to+form+stable+occlusive+thrombi+in+injured+veins.">von Willebrand factor and factor VIII are independently required to form stable occlusive thrombi in injured veins.</a> Blood. 109 (6), 2424-2429 (2007).</li><li>Andre, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40L+stabilizes+arterial+thrombi+by+a+beta3+integrin--dependent+mechanism.">CD40L stabilizes arterial thrombi by a beta3 integrin--dependent mechanism.</a> Nature Medicine. 8 (3), 247-252 (2002).</li><li>Ni, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+platelet+thrombus+formation+in+arterioles+of+mice+lacking+both+von+Willebrand+factor+and+fibrinogen.">Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen.</a> Journal of Clinical Investigation. 106 (3), 385-392 (2000).</li><li>Bergmeier, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+platelet+adhesion+receptor+GPIbalpha+far+exceeds+that+of+its+main+ligand,+von+Willebrand+factor,+in+arterial+thrombosis.">The role of platelet adhesion receptor GPIbalpha far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis.</a> Proceedings of the National Academy of Sciences. 103 (45), 16900-16905 (2006).</li><li>Ciciliano, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+multifaceted+mechanisms+of+the+ferric+chloride+thrombosis+model+using+an+interdisciplinary+microfluidic+approach.">Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach.</a> Blood. 126 (6), 817-824 (2015).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+FeCl3-induced+arterial+thrombosis.">Mechanisms underlying FeCl3-induced arterial thrombosis.</a> Journal of Thrombosis and Haemostasis. 9 (4), 779-789 (2011).</li><li>Woollard, K. J., Sturgeon, S., Chin-Dusting, J. P. F., Salem, H. H., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+hemolysis+and+hemoglobin+oxidation+promote+ferric+chloride-induced+vascular+injury.">Erythrocyte hemolysis and hemoglobin oxidation promote ferric chloride-induced vascular injury.</a> Journal of Biological Chemistry. 284 (19), 13110-13118 (2009).</li><li>Shim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+ferric+chloride-induced+arterial+thrombosis+model+of+mice+and+the+role+of+red+blood+cells+in+thrombosis+acceleration.">Characterization of ferric chloride-induced arterial thrombosis model of mice and the role of red blood cells in thrombosis acceleration.</a> Yonsei Medical Journal. 62 (11), 1032-1041 (2021).</li><li>Ghosh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+prothrombotic+potential+of+four-factor+prothrombin+complex+concentrate+(4F-PCC)+in+animal+models.">Evaluation of the prothrombotic potential of four-factor prothrombin complex concentrate (4F-PCC) in animal models.</a> PLoS One. 16 (10), 0258192 (2021).</li><li>Wilbs, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+peptide+FXII+inhibitor+provides+safe+anticoagulation+in+a+thrombosis+model+and+in+artificial+lungs.">Cyclic peptide FXII inhibitor provides safe anticoagulation in a thrombosis model and in artificial lungs.</a> Nature Communications. 11 (1), 3890 (2020).</li><li>Wei, Y., Deng, X., Sheng, G., Guo, X. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rabbit+model+of+cerebral+venous+sinus+thrombosis+established+by+ferric+chloride+and+thrombin+injection.">A rabbit model of cerebral venous sinus thrombosis established by ferric chloride and thrombin injection.</a> Neuroscience Letters. 662, 205-212 (2018).</li><li>Jacob-Ferreira, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithrombotic+activity+of+Batroxase,+a+metalloprotease+from+Bothrops+atrox+venom,+in+a+model+of+venous+thrombosis.">Antithrombotic activity of Batroxase, a metalloprotease from Bothrops atrox venom, in a model of venous thrombosis.</a> International Journal of Biological Macromolecules. 95, 263-267 (2017).</li><li>Zhou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rabbit+model+of+cerebral+microembolic+signals+for+translational+research:+preclinical+validation+for+aspirin+and+clopidogrel.">A rabbit model of cerebral microembolic signals for translational research: preclinical validation for aspirin and clopidogrel.</a> Journal of Thrombosis and Haemostasis. 14 (9), 1855-1866 (2016).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+evodiamine+on+collagen-induced+platelet+activation+and+thrombosis.">Effect of evodiamine on collagen-induced platelet activation and thrombosis.</a> BioMed Research International. 2022, 4893859 (2022).</li><li>Li, W., McIntyre, T. M., Silverstein, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride-induced+murine+carotid+arterial+injury:+A+model+of+redox+pathology.">Ferric chloride-induced murine carotid arterial injury: A model of redox pathology.</a> Redox Biology. 1 (1), 50-55 (2013).</li><li>Li, W., Nieman, M., Sen Gupta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride-induced+murine+thrombosis+models.">Ferric chloride-induced murine thrombosis models.</a> Journal of Visualized Experiments. (115), e54479 (2016).</li><li>Holly, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ether+lipid+metabolism+by+AADACL1+regulates+platelet+function+and+thrombosis.">Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis.</a> Blood Advances. 3 (22), 3818-3828 (2019).</li><li>Bird, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+the+therapeutic+index+of+marketed+anti-coagulants+and+anti-platelet+agents+by+guinea+pig+models+of+thrombosis+and+hemostasis.">Prediction of the therapeutic index of marketed anti-coagulants and anti-platelet agents by guinea pig models of thrombosis and hemostasis.</a> Thrombosis Research. 123 (1), 146-158 (2008).</li></ol>

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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			<td>Sodium Cacodylate Buffer 0.2M, pH7.4</td>
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			<td>WinDAQ/100 Software for Windows</td>
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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 ...

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

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1.6K Views.  University of Kentucky. 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.

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

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...