This work was supported by a grant from the Heart and Stroke Foundation of Canada and by The Morris and Bella Fainman Family Foundation. The authors would like to thank Veronique Michaud for her technical help with the VEVO770 ultrasound imaging system.

All procedures were approved by the institutional Animal Care Committee of McGill University Montr&#233;al, QC, Canada. All the equipment required is listed in Table I.
1. Murine (C57BL/6J) IVC Stasis Protocol
<ol>
	<li>Surgery preparation 
	NOTE: This is a survival surgery and therefore, proper aseptic technique needs to be followed at all times. This includes making sure all materials are at hand, cleaning and sterilizing instruments before and between use, and design...

<ol>
	<li>Fowkes, F. J., Price, J. F., Fowkes, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+diagnosed+deep+vein+thrombosis+in+the+general+population:+systematic+review.">Incidence of diagnosed deep vein thrombosis in the general population: systematic review.</a> Eur J Vasc Endovasc Surg. 25 (1), 1-5 (2003).</li><li>McGuinness, C. 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=Recruitment+of+labelled+monocytes+by+experimental+venous+thrombi.">Recruitment of labelled monocytes by experimental venous thrombi.</a> Thromb Haemost. 85 (6), 1018-1024 (2001).</li><li>Singh, I., 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=Failure+of+thrombus+to+resolve+in+urokinase-type+plasminogen+activator+gene-knockout+mice:+rescue+by+normal+bone+marrow-derived+cells.">Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow-derived cells.</a> Circulation. 107 (6), 869-875 (2003).</li><li>Itoh, K., Ieko, M., Hiraguchi, E., Kitayama, H., Tsukamoto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+kinetics+of+99mTc+labeled+recombinant+tissue+plasminogen+activator+in+rabbits.">In vivo kinetics of 99mTc labeled recombinant tissue plasminogen activator in rabbits.</a> Ann Nucl Med. 8 (3), 193-199 (1994).</li><li>Kang, C., Bonneau, M., Brouland, J. P., Bal dit Sollier, C., Drouet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+pig+models+of+venous+thrombosis+mimicking+human+disease.">In vivo pig models of venous thrombosis mimicking human disease.</a> Thromb Haemost. 89 (2), 256-263 (2003).</li><li>Knight, L. C., Baidoo, K. E., Romano, J. E., Gabriel, J. L., Maurer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pulmonary+emboli+and+deep+venous+thrombi+with+99mTc-bitistatin,+a+platelet-binding+polypeptide+from+viper+venom.">Imaging pulmonary emboli and deep venous thrombi with 99mTc-bitistatin, a platelet-binding polypeptide from viper venom.</a> J Nucl Med. 41 (6), 1056-1064 (2000).</li><li>Wakefield, T. 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=Venous+thrombosis+prophylaxis+by+inflammatory+inhibition+without+anticoagulation+therapy.">Venous thrombosis prophylaxis by inflammatory inhibition without anticoagulation therapy.</a> J Vasc Surg. 31 (2), 309-324 (2000).</li><li>Robins, R. 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=Vascular+Gas6+contributes+to+thrombogenesis+and+promotes+tissue+factor+up-regulation+after+vessel+injury+in+mice.">Vascular Gas6 contributes to thrombogenesis and promotes tissue factor up-regulation after vessel injury in mice.</a> Blood. 121 (4), 692-699 (2013).</li><li>Aghourian, M. N., Lemarie, C. A., Bertin, F. R., Blostein, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E+synthase+is+upregulated+by+Gas6+during+cancer-induced+venous+thrombosis.">Prostaglandin E synthase is upregulated by Gas6 during cancer-induced venous thrombosis.</a> Blood. 127 (6), 769-777 (2016).</li><li>Laurance, 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=Gas6+(Growth+Arrest-Specific+6)+Promotes+Inflammatory+(CCR2hiCX3CR1lo)+Monocyte+Recruitment+in+Venous+Thrombosis.">Gas6 (Growth Arrest-Specific 6) Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.</a> Arterioscler Thromb Vasc Biol. , (2017).</li><li>Diaz, J. 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=Critical+review+of+mouse+models+of+venous+thrombosis.">Critical review of mouse models of venous thrombosis.</a> Arterioscler Thromb Vasc Biol. 32 (3), 556-562 (2012).</li><li>Aghourian, M. N., Lemarie, C. A., Blostein, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+monitoring+of+venous+thrombosis+in+mice.">In vivo monitoring of venous thrombosis in mice.</a> J Thromb Haemost. 10 (3), 447-452 (2012).</li><li>Brandt, M., 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=Deep+vein+thrombus+formation+induced+by+flow+reduction+in+mice+is+determined+by+venous+side+branches.">Deep vein thrombus formation induced by flow reduction in mice is determined by venous side branches.</a> Clin Hemorheol Microcirc. 56 (2), 145-152 (2014).</li><li>Diaz, J. A., Farris, D. M., Wrobleski, S. K., Myers, D. D., Wakefield, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferior+vena+cava+branch+variations+in+C57BL/6+mice+have+an+impact+on+thrombus+size+in+an+IVC+ligation+(stasis)+model.">Inferior vena cava branch variations in C57BL/6 mice have an impact on thrombus size in an IVC ligation (stasis) model.</a> J Thromb Haemost. 13 (4), 660-664 (2015).</li><li>Luther, N., 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=Innate+Effector-Memory+T+Cell+Activation+Regulates+Post-Thrombotic+Vein+Wall+Inflammation+and+Thrombus+Resolution.">Innate Effector-Memory T Cell Activation Regulates Post-Thrombotic Vein Wall Inflammation and Thrombus Resolution.</a> Circ Res. , (2016).</li><li>Subramaniam, 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=Distinct+contributions+of+complement+factors+to+platelet+activation+and+fibrin+formation+in+venous+thrombus+development.">Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development.</a> Blood. 129 (16), 2291-2302 (2017).</li><li>Diaz, J. 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=Thrombogenesis+with+continuous+blood+flow+in+the+inferior+vena+cava.+A+novel+mouse+model.">Thrombogenesis with continuous blood flow in the inferior vena cava. A novel mouse model.</a> Thromb Haemost. 104 (2), 366-375 (2010).</li></ol>

There are several critical steps for successful venous thrombus formation using the stasis model. Induction of vein thrombosis is more challenging in old mice due to the accumulation of fat surrounding the inferior vena cava and the aorta. Ideally, mice undergoing this procedure should be 8 - 10-weeks-old. Great care should be taken not to induce endothelial damage in the IVC during the blunt dissection and ligature. In addition, it is crucial to keep the animal in a 34 &#176;C incubator for at least 30 minutes after the...

Venous thromboembolism (VTE), which is comprised of deep venous thrombosis (DVT) and pulmonary embolism, is the third leading cause of cardiovascular death after myocardial infarction and stroke. It is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 5001. VTE can lead to: 1) death through pulmonary embolism; 2) post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration; or 3) chronic pulmonary hypertension resulting in significant chronic respiratory compromise. VTE is a multi-factorial disease and may result from stasis of blood flow, damage to vessel walls, and/or hypercoagulable states due to a disruption of the balance between the coagulation and the fibrinolytic systems, as it has been described over a hundred years ago by Virchow and is known as the Virchow&#39;s Triad.
Because in most cases it is impossible to obtain human DVT samples, researchers have developed experimental animal models of DVT. Several animals including rat2, mouse3, rabbit4, pig5, dog6, and non-human primate7 have been used. Mice can be genetically modified and are the most frequently used animal to study DVT. However, as in all animals, spontaneous DVT is not observed in mice. Thus, physical or chemical alterations of the vein wall are used to create thrombosis in mice. We have previously used the ferric chloride model to induce thrombosis in the inferior vena cava (IVC) of mice8,9,10. This model has the advantage of reliably producing occlusive thrombi within minutes and can be used to investigate the role of anti-coagulant and anti-platelet drugs during acute DVT. However, it is a terminal procedure. Thus, to study acute and chronic DVT, the stasis model is more suitable. In this model, thrombus formation is induced by the complete interruption of blood flow in the IVC, one of the factors in Virchow&#39;s triad for DVT development. This model can be used to study DVT formation and resolution, which is an advantage compared to the FeCl3 model11.
We have developed a non-invasive method to follow thrombus formation and resolution over time using a micro-imaging high-frequency ultrasound system12. We have previously demonstrated that measurement of venous thrombosis by ultrasound correlates favorably with thrombi obtained pathologically. We have confirmed in two subsequent studies that measurements obtained with ultrasound correlate with thrombus weight and thrombus area quantified by histochemistry9,10. More importantly, we have showed that high frequency ultrasound can be used to monitor the formation of deep venous thrombosis in mice12. It may also be used to quantify thrombus resolution in a non-invasive way.
Here, we will describe the protocol allowing thrombus formation using the stasis-induced thrombosis mouse model and how thrombus formation can be monitored non-invasively over time using high frequency ultrasound.

<table>
	<tbody>
		<tr>
			<td>6-0 perma-hand silk suture</td>
			<td>Ethicon</td>
			<td>706G</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>20830-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>20830-00</td>
			<td></td>
		</tr>
		<tr>
			<td>blunt forceps (straight and curved)</td>
			<td>Fine Science Tools</td>
			<td>20830-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Driver</td>
			<td>Fine Science Tools</td>
			<td>13002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15396-00</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml syringes</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles</td>
			<td>Becton Dickinson &#38; Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VEVO 770 High Resolution Imaging System</td>
			<td>Visualsonics</td>
			<td>No longer sold</td>
			<td></td>
		</tr>
		<tr>
			<td>SR Buprenorphine</td>
			<td>ZooPharm</td>
			<td></td>
			<td>Given to LDI by Vet</td>
		</tr>
		<tr>
			<td>Surgery Microscope</td>
			<td>Leica</td>
			<td>Leica M651</td>
			<td></td>
		</tr>
		<tr>
			<td>Systan eye oinment</td>
			<td>Alcon</td>
			<td>288/28062-0</td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 sterile Gauze</td>
			<td>CDMV</td>
			<td>#104148</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tip Applicators</td>
			<td></td>
			<td></td>
			<td>from JGH</td>
		</tr>
		<tr>
			<td>Transpore hypoallergenic surgical tape</td>
			<td>CDMV</td>
			<td>#7411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel (Aquasonic-100)</td>
			<td>Dufort &#38; Lavigne</td>
			<td>#AKEN4061</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td>From JGH</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Dispomed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic chamber,hoses, and adminstration equipment</td>
			<td>Dispomed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hair remover</td>
			<td>Nair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water heated hard pad</td>
			<td>Braintree Scientific, Inc.</td>
			<td>#HHP-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaymar heater water pump TP500</td>
			<td>MATVET Inc.</td>
			<td>#R-500305</td>
			<td></td>
		</tr>
		<tr>
			<td>Infra-red heating lamp</td>
			<td>electrimat inc.</td>
			<td>#1R175R-PAR</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse rectal temperature prope</td>
			<td>emkaTECHNOLOGIES</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td></td>
			<td></td>
			<td>From JGH</td>
		</tr>
	</tbody>
</table>

deep vein thrombosis induced by stasis in mice monitored by high frequency ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

Stasis venous thrombosis model
In the stasis model, mice are anesthetized, and an incision is made to expose the inferior vena cava (IVC). The incision is made on the left or right side of the mouse instead of a midline laparotomy in a way that would not interfere with the ultrasound probe. The abdominal muscles and the skin are fold back to expose the IVC (Figure 1). First, side br...

Watch this Scientific Journal Video about Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography at JoVE.com

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

The overall goals of this procedure are to induce deep vein thrombosis in mice using the inferior vena cava stasis model, and to monitor the thrombi using high frequency ultrasound. This method can help answer key questions in the venous thrombosis field about how a pharmacological agent, gene, or cell type of interest can influence thrombus formation or resolution. The main advantage of this technique is that both thrombus formation and resolution can be non-invasively quantified in mice.We first had the idea of this method when we were investigating how to denamicate and non-invasively monitor thrombus formation in the inferior vena cava of mice. To begin, use forceps to lift the lower abdominal skin of an eight to 10 week old C57 black six mouse. And use surgical scissors to make a vertical incision parallel to either side of the linea alba from the midline.Make a horizontal incision at the top of the abdomen. Followed by repeat incisions through the abdominal muscle layer. Fold the skin and muscle away from the incision to expose the abdominal cavity.And place moistened gauze onto both sides of the wound. Apply gentle pressure to both sides of the abdomen to externalize the intestines onto the gauze. And place a second layer of moistened gauze over the externalized tissue.Under a dissecting microscope, move aside any peritoneal fat to expose the interior vena cava, or IVC, between the renal and iliac veins. And locate the IVC side branches. Using forceps, gently blunt dissect around each side branch to break through the fascia without damaging the surrounding vessels.Next, carefully grasp the fat around one branch to lift the vein. And pass a piece of six dash zero silk suture under the vessel. Then use suture forceps and a standard surgical knot tying technique to make a surgical ligation with at least three throws around the side branch to completely occlude the the vessel.Alternatively, use Hemoclips. To separate the aorta from the IVC, blunt dissect around and between both vessels immediately distal from the left renal vein without damaging either vessel. When a clear window has been created between the vessels, pass a section of six dash zero silk suture under the IVC and the abdominal aorta.And use forceps to pull the suture through the window. Using suture forceps, ligate the IVC with three throws immediately distal to the left renal vein for full occlusion of the vessel. Confirm that the abdominal aorta has been left uninterrupted.And that all of the side branches have been ligated. The IVC will appear dilated distal from the ligation site. And no blood flow will be visible.Return the peritoneal fat and the intestines to the abdominal cavity. And use suture forceps and six dash zero silk sutures to separately close the muscle and skin layers with running stitches. Then place the animal in a 34 degree Celsius incubator for at least 30 minutes with monitoring until full recovery.Place the ultrasound gel bottle through the coils of a water heating system to heat the gel to 37 degrees Celsius. 24 hours after the ligation, place the animal on the analysis platform. And apply electrode gel onto the four electrodes of the platform.With the animal in the supine position, fix the paws to the electrodes. And insert a lubricated thermometer into the rectum. Place gauze to the side of the animal to catch any excess ultrasound gel.Then apply the gel to the exposed skin. And image the IVC according to the standard ultrasound imaging protocols. Using a high frequency microimaging system, the IVC can be identified in the longitudinal view prior to ligation.And the flow velocity can be determined by Pulsed wave Doppler imaging. Immediately after the ligation, a decrease in the blood flow is apparent. 24 hours after the ligation, dense thrombi can be visualized inside the IVC by ultrasonography.Ultrasonography also allows quantification of the velocity of blood flow in the vessels. Using colored Doppler before, immediately after, and 24 hours after ligation. Once mastered, the thrombus can be induced in 30 to 45 minutes depending on the presence and number of side branches if the technique is performed properly.While attempting this procedure, it's important to remember to take your time and to be precise yet gentle in your movements as it is relatively easy to rupture the vessels. Following this procedure, other methods like immunofluorescent, histological, or flow cytometric analysis can be performed to answer additional questions about the cellular content of thrombi or to quantify specific proteins of interest.

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Immunology and Infection

9.7K visualizações.  McGill University. The overall goals of this procedure are to induce deep vein thrombosis in mice using the inferior vena cava stasis model, and to monitor the thrombi using high frequency ultrasound. This method can help answer key questions in the venous thrombosis field about how a pharmacological agent, gene, or cell type of interest can influence thrombus formation or resolution. The main advantage of this technique is that both thrombus formation and resolution can be non-invasively quantified in mice.