Name: ferric chloride-induced arterial thrombosis and sample collection for 3d electron microscopy analysis
Uploaded: 2023-03-17T13:45:00
Description: Watch this Scientific Journal Video about Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis at JoVE.com

The authors thank the members of the Whiteheart Laboratory for their careful perusal of this manuscript. The work was supported by grants from the NIH, NHLBI (HL56652, HL138179, and HL150818), and a Department of Veterans Affairs Merit Award to S.W.W., R01 HL 155519 to B.S., and NIBIB intramural program grant to R.D.L.

All experiments discussed here were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky.
NOTE: Surgical instruments are listed in Figure 1 and the Table of Materials. C57BL/6J mice, 8-10 weeks old, male/female or relevant genetically manipulated (Knockout or Knockin) strains were used.
1. FeCl3-induced carotid artery injury
<ol>
	<l...

<ol>
	<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 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Saline&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP358-212</td>
			<td>NaCl used to make a solution of 0.9% saline&#160;</td>
		</tr>
		<tr>
			<td>1 mL Syringe&#160;</td>
			<td>Becton, Dickinson and Company&#160;</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>190 Proof Ethanol&#160;</td>
			<td>KOPTEC</td>
			<td>V1101&#160;</td>
			<td>Used to make a 70% ethanol solution to use for prepping the mouse for surgery&#160;</td>
		</tr>
		<tr>
			<td>2,2,2 Tribromoethanol</td>
			<td>Sigma Aldrich</td>
			<td>48402</td>
			<td></td>
		</tr>
		<tr>
			<td>25 Yard Black Braided Silk Suture (5-0)</td>
			<td>DEKNATEL</td>
			<td>136082-1204</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x 3/8 Needle&#160;</td>
			<td>Becton, Dickinson and Company&#160;</td>
			<td>305110</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methyl-2-butanol</td>
			<td>Sigma Aldrich</td>
			<td>240486</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5 mL Transfer Pipet, Graduated to 3 mL</td>
			<td>Globe Scientific Inc.</td>
			<td>135010</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep Pads (70% Isopropyl Alcohol)</td>
			<td>Medline</td>
			<td>MDS090735</td>
			<td></td>
		</tr>
		<tr>
			<td>Araldite GY 502&#160;</td>
			<td>Electron microscopy Services&#160;</td>
			<td>10900</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish 35mm X 10mm&#160;</td>
			<td>Corning Incorporated&#160;</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Scale&#160;</td>
			<td>Ward&#39;s Science&#160;</td>
			<td>470314-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors, 12.5 cm long</td>
			<td>World Precision Instrument</td>
			<td>15922-G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30 activator&#160;</td>
			<td>Electron microscopy Services&#160;</td>
			<td>13600</td>
			<td></td>
		</tr>
		<tr>
			<td>Dodenyl Succinic Anhydride/ DDSA</td>
			<td>Electron microscopy Services&#160;</td>
			<td>13700</td>
			<td></td>
		</tr>
		<tr>
			<td>Doggy Poo Bags/animal carcass disposal bag</td>
			<td>Crown Products&#160;</td>
			<td>PP-RB-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Doppler FlowProbe</td>
			<td>Transonic Systems Inc.</td>
			<td>MA0.5PSB</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBED 812 resin&#160;</td>
			<td>Electron microscopy Services&#160;</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol, anhydrous 200 proof&#160;</td>
			<td>Electron microscopy Services&#160;</td>
			<td>15055</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Dressing Forceps, 4&#34; Full Curved, Standard, 0.8mm Wide Tips</td>
			<td>Integra Miltex</td>
			<td>18-784</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper&#160;</td>
			<td>VWR</td>
			<td>28310-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp-Blunt</td>
			<td>Fine Science Tools&#160;</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Finger Loop Ear Punches&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>24212-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Sponges 2&#8221; x 2&#8221; &#8211; 12 Ply&#160;</td>
			<td>Dukal Corporation</td>
			<td>2128</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (10% solution)</td>
			<td>Electron microscopy Services&#160;</td>
			<td>16120</td>
			<td></td>
		</tr>
		<tr>
			<td>Integra Miltex Carbon Steel Surgical Blade #10</td>
			<td>Integra&#174; Miltex&#174;</td>
			<td>4110</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (III) Chloride&#160;</td>
			<td>SIGMA-ALDRICH</td>
			<td>157740-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Knife Handle Miltex&#174; Extra Fine Stainless Steel Size 3</td>
			<td>Integra Lifesciences&#160;</td>
			<td>157510</td>
			<td></td>
		</tr>
		<tr>
			<td>L-aspartic acid</td>
			<td>Sigma&#160;Fisher</td>
			<td>&#160;A93100</td>
			<td></td>
		</tr>
		<tr>
			<td>L-aspartic acid</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP374-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead Nitrate&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>L-62</td>
			<td></td>
		</tr>
		<tr>
			<td>LEICA S8AP0 Microscope</td>
			<td>LEICA</td>
			<td>No longer available</td>
			<td>No longer available from the company</td>
		</tr>
		<tr>
			<td>LEICA S8AP0 Microscope Stand&#160;</td>
			<td>LEICA</td>
			<td>10447255</td>
			<td>No longer available from the company</td>
		</tr>
		<tr>
			<td>Light-Duty Tissue Wipers&#160;</td>
			<td>VWR</td>
			<td>82003-822</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Forceps; 1x2 Teeth, Full Curve; 0.8 mm Tip Width; 4&#34; Length</td>
			<td>Roboz Surgical Instrument Company</td>
			<td>RS-5157</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide 4% aqueous solution&#160;</td>
			<td>Electron microscopy Services&#160;</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% solution)</td>
			<td>Electron microscopy Services&#160;</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferricyanide</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-8131</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene Oxide, ACS reagent&#160;</td>
			<td>Electron microscopy Services&#160;</td>
			<td>20401</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainin Classic Pipette PR-10</td>
			<td>Rainin</td>
			<td>17008649</td>
			<td></td>
		</tr>
		<tr>
			<td>Research Flowmeter&#160;</td>
			<td>Transonic Systems Inc.</td>
			<td>T402B01481</td>
			<td>Model: T402</td>
		</tr>
		<tr>
			<td>Scotch Magic Invisible Tape, 3/4&#34; x 1000&#34;, Clear</td>
			<td>Scotch&#160;</td>
			<td>305289</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Heated Pad</td>
			<td>K&#38;H Manufacturing Inc.</td>
			<td>Model: HM10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate Buffer 0.2M, pH7.4</td>
			<td>Electron microscopy Services&#160;</td>
			<td>11623</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cotton Tipped Applicators&#160;</td>
			<td>Puritan Medical Products&#160;</td>
			<td>25-806 1WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Steromaster Illuminator&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-562-21</td>
			<td>No longer available from the company</td>
		</tr>
		<tr>
			<td>Surgical Dumont #7 Forceps&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide (TCH)</td>
			<td>SIGMA-ALDRICH</td>
			<td>88535</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Low Retention Pipet Tip Reloads (0.1-10 &#181;L)</td>
			<td>VWR</td>
			<td>76323-394</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron microscopy Services&#160;</td>
			<td>22400</td>
			<td></td>
		</tr>
		<tr>
			<td>Veet Gel Cream Hair Remover</td>
			<td>Reckitt Benckiser</td>
			<td>3116875</td>
			<td></td>
		</tr>
		<tr>
			<td>White Antistatic Hexagonal Weigh Boats, Medium, 64 x 15 x 19 mm</td>
			<td>Fisher Scientific&#160;</td>
			<td>S38975</td>
			<td></td>
		</tr>
		<tr>
			<td>WinDAQ/100 Software for Windows</td>
			<td>DATAQ Instruments, Inc.</td>
			<td>Version 3.38</td>
			<td>Freely available to download. https://www.dataq.com/products/windaq/</td>
		</tr>
		<tr>
			<td>ZEISS AxioCam Icc 1</td>
			<td>ZEISS</td>
			<td>57615</td>
			<td></td>
		</tr>
	</tbody>
</table>

ferric chloride-induced arterial thrombosis and sample collection for 3d electron microscopy analysis

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

The data are generally presented as time to occlusion, or time required to form a fully occlusive thrombus. These data can be plotted as a Kaplan-Meier survival curve (Figure 4A)19, a dot plot with bars showing the terminal blood flow at the time of either cessation of the blood flow or the termination of an experiment (Figure 4B), or as a line graph (Figure 4C). Thrombus stability can be studied using this t...

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

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

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like ...

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

Research

JoVE Journal

Medicine

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 ...

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 ...

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 ...

Ferric Chloride-induced Murine Thrombosis Models

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

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

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 ...

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

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 ...

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 ...

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, ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...