This work was supported by the British Heart Foundation&#160;(PG/13/60/30406) and the University of Birmingham.

All animal procedures were approved by Animal Welfare Ethical Review Body and the UK Home Office (UK, Project License 40/3745). Mice on C57BL/6 background, 8 - 12 week old, 19 - 25 g body weight, of both genders are used.
1. Animal Anesthesia
<ol>
	<li>Place a mouse into an induction chamber and induce anesthesia by a mixture of 5% isoflurane with 100% oxygen. Use a flow rate of 0.5 L/min. Wait until the mouse completely stops moving and its frequency of breathing becomes rare.</li>
	<li>Remove the mouse from the chamber and shave the abdomen with an electric clipper. Remove hair from the abdomen as thoroughly as possible to avoid contamination of the abdominal cavity.</li>
	<li>Place the mouse in the supine position and place mouse&#39;s nose into a cone linked to the anesthesia system. Decrease the percent of isoflurane to 2-3% (the exact percent may slightly differ from animal to animal). Make sure the animal is sleeping with its respiratory rate being lower than usual but avoiding gasping. If gasping occurs, decrease the percent of isoflurane by 0.5%.
	<ol>
		<li>Check the pedal reflex to confirm proper anesthetization and start surgery only if it is negative. Apply vet ointment&#160;on the eyes to prevent dryness.</li>
	</ol>
	</li>
	<li>Apply an anti-bactericidal solution (1% Hibitane) on the mouse&#39;s abdomen and wipe the area using a sterile cotton bud in a way excluding potential re-contamination of the surgical site (e.g., inside to out in a circular fashion). Repeat 3 times.</li>
</ol>
2. Application of the IVC Stenosis and Mouse Recovery
NOTE: All the instruments as well as materials (cotton buds, gauze, saline etc.) coming into contact with the surgery area must be sterile. The operator must scrub up prior to surgery and wear sterile gloves and gown during the procedure. Microscope handles should be wrapped in a sterile foil.
<ol>
	<li>Give mice an anti-pain treatment prior to surgery and during the entire experimental period. For example, use buprenorphine 0.1 mg/kg subcutaneously 30 min before surgery and then every 12 hours until the animal is euthanized. As thrombosis in this method develops similarly to sterile inflammation, avoid using anti-inflammatory drugs as a premedication.</li>
	<li>Cover the mouse with a sterile drape and make a hole in the drape to expose the site of surgery. Using scissors, make an incision along the abdominal midline, 1.5 cm down from the sternum. Using cotton buds exteriorize the intestines to the left-hand side of the mouse and cover them with sterile gauze soaked in warm (37 &#176;C) saline to avoid drying out.</li>
	<li>Identify the IVC and its side branches (there may be 0, 1 or 2 of them) in the caudal direction from the site of fusion of the IVC with the left renal vein. Ligate all visible side branches with 7-0 inert Prolene sutures as close to the IVC as possible.
	<ol>
		<li>Try not to hold the side branches with the forceps directly but rather pull them up holding the fat tissue surrounding them with forceps in one hand and make a tunnel beneath the branch with forceps in the other hand. Do not close back branches.</li>
	</ol>
	</li>
	<li>Holding surrounding tissues with forceps, pull the IVC up and left producing tension in the small area between the IVC and the aorta exactly at the angle between the IVC and the left renal vein. Take into account that it is virtually impossible to separate the aorta from the IVC even 2-3 mm below (in the caudal direction).</li>
	<li>Using diverging movements with forceps tips in your right hand make a hole between the aorta and the IVC. Keep in mind that the more movements made, the higher the chance of damaging the vessel. Ideally, try to minimize the number of movements to 3-5.</li>
	<li>Prepare a piece of 7-0 inert Prolene suture of about 2 cm long. Place it in the mouse&#39;s peritoneal cavity on the operator&#39;s left-hand side in the vicinity to the IVC.</li>
	<li>Pull the IVC up and left again. Insert your right forceps tips into the hole between the aorta and the IVC so that the tips appear on the other side of the IVC. Take the suture and pull it back through the hole (Figure 3A).</li>
	<li>Make a provisional knot with the suture but do not close it. Place 30G needle (&#34;a spacer&#34;) bent as a hook into the knot and now close it (Figure 3B).</li>
	<li>Remove the spacer (Figure 3C).</li>
	<li>Return the intestines back to the peritoneal cavity with a cotton bud and distribute them equally in it.</li>
	<li>Close peritoneum with 6-0 vicryl suture using continuous loops.Close the first and last loops as a knot.</li>
	<li>Close skin using metal staples.</li>
	<li>Inject the mouse with 0.5 mL of warm (37 &#176;C) glucose saline subcutaneously to restore the liquid balance of the organism.</li>
	<li>Place the mouse into an individual cage in a warm (25 &#176;C) chamber or room and put food and gel water inside the cage. Observe until the mouse is fully recovered (generally, around 1 h).</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secular+trends+in+occurrence+of+acute+venous+thromboembolism:+the+Worcester+VTE+study+(1985-2009).">Secular trends in occurrence of acute venous thromboembolism: the Worcester VTE study (1985-2009).</a> Am J Med. 127 (9), 829-839 (2014).</li><li>House of Commons Health Care Committee. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prevention+of+Venous+Thromboembolism+in+Hospitalised+patients.">The Prevention of Venous Thromboembolism in Hospitalised patients.</a> The House of Commons. , (2005).</li><li>Kuipers, 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=Travel+and+venous+thrombosis:+a+systematic+review.">Travel and venous thrombosis: a systematic review.</a> J Intern Med. 262 (6), 615-634 (2007).</li><li>Lopez, J. 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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=Monocytes,+neutrophils,+and+platelets+cooperate+to+initiate+and+propagate+venous+thrombosis+in+mice+in+vivo.">Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.</a> J Exp Med. 209 (4), 819-835 (2012).</li><li>Sevitt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+growth+of+valve-pocket+thrombi+in+femoral+veins.">The structure and growth of valve-pocket thrombi in femoral veins.</a> J Clin Pathol. 27 (7), 517-528 (1974).</li><li>Witsch, T., 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+novel+hollow+and+perforated+flexible+wire+allows+the+safe+and+effective+local+application+of+thrombolytic+therapy+in+a+mouse+model+of+deep+vein+thrombosis.">A novel hollow and perforated flexible wire allows the safe and effective local application of thrombolytic therapy in a mouse model of deep vein thrombosis.</a> J Thromb Thrombolysis. 37 (4), 450-454 (2014).</li><li>Shaya, S. 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=Comparison+of+the+effect+of+dabigatran+and+dalteparin+on+thrombus+stability+in+a+murine+model+of+venous+thromboembolism.">Comparison of the effect of dabigatran and dalteparin on thrombus stability in a murine model of venous thromboembolism.</a> J Thromb Haemost. 14 (1), 143-152 (2016).</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> J Clin Invest. 106 (3), 385-392 (2000).</li><li>Wrobleski, S. K., Farris, D. M., Diaz, J. A., Myers, D. D., Wakefield, T. 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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=Macrovascular+thrombosis+is+driven+by+tissue+factor+derived+primarily+from+the+blood+vessel+wall.">Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall.</a> Blood. 105 (1), 192-198 (2005).</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=Impaired+fibrinolytic+system+in+ApoE+gene-deleted+mice+with+hyperlipidemia+augments+deep+vein+thrombosis.">Impaired fibrinolytic system in ApoE gene-deleted mice with hyperlipidemia augments deep vein thrombosis.</a> J Vasc Surg. 55 (3), 815-822 (2012).</li><li>Zhou, J., May, L., Liao, P., Gross, P. L., Weitz, J. I. <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+ligation+rapidly+induces+tissue+factor+expression+and+venous+thrombosis+in+rats.">Inferior vena cava ligation rapidly induces tissue factor expression and venous thrombosis in rats.</a> Arterioscler Thromb Vasc Biol. 29 (6), 863-869 (2009).</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=Electrolytic+inferior+vena+cava+model+(EIM)+of+venous+thrombosis.">Electrolytic inferior vena cava model (EIM) of venous thrombosis.</a> J Vis Exp. (53), e2737 (2011).</li><li>Diaz, J. 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The authors have nothing to disclose.

Here, a protocol of the IVC stenosis, which mimics blood flow distortion, a major triggering factor for DVT, is presented. Stenosis of the IVC induces development of thrombi in 65-100% of C57BL/6 mice within 48 h and 25-50% of mice within 2-6 h6,8,23 (Brill A, unpublished data, 2016). A major limitation of the method is the variability in thrombus size24 (Figure 4), which is observed after both short-term (6h) and long-term (48 h) IVC stenosis. The reasons for such variability (given that the same conditions, such as the spacer, anesthesia etc., are used) remain obscure but one may speculate that anatomical variations between the mice (e.g., width of the IVC, number and location of both side and back branches) underlie this phenomenon. The variability in thrombus size makes thrombosis prevalence (percent of mice with a thrombus) the main outcome. Thrombosis prevalence can be compared using a contingency table and the Fisher&#39;s exact test. One of the experiments was an exception when reduced thrombus size with the same thrombosis prevalence after injection of podoplanin-inhibiting antibodies was observed 7.
This method is especially useful when venous thrombosis initiation is studied. It allows for investigating early events in the vessel wall, such as recruitment of cells, leading eventually to thrombosis. Pro- or anti-thrombotic effects of drugs can be evaluated using this model with thrombosis prevalence being a primary readout. Stenosis for 48 h is applicable to reveal an anti-thrombotic phenotype, whereas 6 h stenosis can be used if a prothrombotic phenotype is expected. Histological analysis of the thrombus and surrounding IVC wall can also be performed.
The question whether side branches should be ligated or left patent remains open. One group has shown that ligation of side branches does not increase thrombus size and location of a side branch closer than 1.5 mm to the IVC ligation site dramatically impairs thrombus development25. Closure of side branches may induce endothelial injury in them and also increase the time of surgery26. In our hands, lack of side branch closure substantially decreases thrombosis prevalence (down to 10 - 30% after 48 h stenosis; Brill, unpublished) and therefore we ligate all visible side branches.
Ideally, littermate controls should be used as mice, even on the same background but from different sources, may have slightly different thrombosis prevalence. If the effect of DVT itself on any parameters (for example, biochemical) is studied, sham-operated animals should be used. Sham-operated mice undergo the same procedure but the ligature around the IVC is closed loosely and left there without producing stenosis.
The most frequent mistake (a critical step) in this protocol is an effort to separate the aorta and the IVC not precisely at the angle between the vessels but slightly lower, which usually results in bleeding. When a massive bleeding occurs, good recovery of the mouse becomes unlikely and it is recommended to stop the experiment and euthanize the animal. Normally, mice recover well, move inside the cage and do not substantially lose weight. We recommend using mice above 20 g for both males and females and keep animals (especially males) in individual cages after surgery until the end of the experiment to avoid fighting and injury. It has been reported in another (electrolytic) model of DVT that male mice produce larger thrombi than females27. Analysis of our data has not revealed substantial difference in thrombosis prevalence between male and female mice (Brill, unpublished). Therefore, researchers are encouraged to perform experiments using the IVC stenosis model on mice of both genders.
Unravelling of sutures and/or staples may not be ruled out especially in long experiments (a week and longer). Thus, mice should be checked at least twice a day specifically for suture integrity and a special attention should be given to the appearance of blood traces on the cage bedding.
It should be noted that, like any other animal model, stenosis of the IVC has its limitations in terms of translation into humans. For example, murine IVCs do not have valves, whereas human DVT develops inside venous valves. Also, humans have vertical spinal orientation with the muscle pump being an important mechanism accelerating blood flow in veins. In contrast, mice have horizontal spinal orientation with no role of the muscle pump in supporting blood return to the heart. These limitations should be considered when translating mouse data to the human disease.

Deep vein thrombosis (DVT) is the development of thrombi in deep veins, usually (but not only) in legs. In conjunctions with pulmonary embolism (PE; designated together as Venous Thromboembolism, VTE) it develops in approximately 900,000 Americans annually and represents a serious health issue and economical problem1,2. PE, a complication of DVT, occurs when a thrombus gets detached from its initial location and reaches the lungs, which may cause respiratory insufficiency and death. The death toll from VTE exceeds mortality from AIDS, breast cancer and traffic accidents, combined3.
The major factor causing DVT besides known reasons, such as cancer or trauma, is the lack of mobility 4,5. This may result from surgery (especially orthopedic), paralysis, long-haul flights or other reasons. Blood flow in veins depends on the muscle pump and therefore limb immobilization results in stagnant blood flow in venous valves, which leads to thrombosis. The purpose of the method described herein is to recapitulate such blood flow distortion6,7. Partial flow restriction in the inferior vena cava (IVC) mimics conditions created in human venous valves and results in the formation of a thrombus similar in structure to human thrombi6. The large part of a thrombus is red and consists of red blood cells, fibrin and incorporation of platelets. Thrombi have a small &#34;white part&#34; enriched in platelets (Figure 1), which resemble &#34;lines of Zahn&#34; described in human venous thrombi. Both parts of the thrombus contain also neutrophils8, which are among the first cells to be recruited at the site of future thrombus6,9. Neutrophils in the red part expel neutrophil extracellular traps (NETs), whereas neutrophils in the white part seem to be devoid of NETs8. Similarly to human DVT, thrombosis in mice is accompanied by elevated plasma D-dimer levels (Figure 2). Low molecular weight heparin (enoxaparin), used for DVT prophylaxis in patients, also prevents thrombosis in mice. An important advantage of this method is absence of endothelial denudation9, which is characteristic of human DVT10. This feature makes IVC stenosis a more clinically relevant model of DVT than, for example, induction of thrombosis by ferric chloride, which induces endothelial denudation and in which thrombi consist predominantly of platelets11,12,13. A model of complete stasis in the IVC is preferred by several teams14,15,16. In contrast to stenosis, in which residual flow is maintained in the vessel, application of stasis completely stops the flow and thus limits the accessibility of systemically administered substances to the thrombosis site. Also, it seems that mechanisms underlying thrombosis induced by stenosis and stasis are different: stenosis results in the development of local inflammation (activation of the endothelium, release of Weibel-Palade body content, recruitment of immune cells and platelets to the vessel wall) causing &#34;immunothrombosis&#34;6,9,17, whereas stasis seems to induce rather thrombosis based, in particular, on tissue factor and other coagulation and fibrinolysis-related mechanisms18,19,20. Thus, the stenosis and stasis models reflect slightly different aspects of venous thrombus development, although their mechanisms may certainly overlap. Electrolytic IVC model (EIM) of DVT21,22 induces a thrombus only partially occluding the vessel wall. Therefore, it is convenient for testing the effects of systemic administration of different drugs on thrombus growth. This model, however, assumes disruption of the IVC wall integrity (insertion of a needle) and induction of thrombosis by electric current making the pathophysiological relevance of this mechanism at least disputable.

<table style="border-collapse:collapse;width:536pt" width="715">
	<colgroup>
		<col style="width:162pt" width="216" />
		<col style="width:160pt" width="213" />
		<col style="width:89pt" width="119" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">C57BL/6 mice</td>
			<td class="xl63" style="width:160pt" width="213">Charles River</td>
			<td class="xl63" style="width:89pt" width="119"></td>
			<td class="xl66" style="width:125pt" width="167">8 - 10 weeks old, bothe genders</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Scissors</td>
			<td class="xl63" style="width:160pt" width="213">WPI</td>
			<td align="right" class="xl63" style="width:89pt" width="119">15922</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Scissors</td>
			<td class="xl63" style="width:160pt" width="213">WPI</td>
			<td align="right" class="xl63" style="width:89pt" width="119">14003</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Dumont 5/45 forceps</td>
			<td class="xl63" style="width:160pt" width="213">FST</td>
			<td class="xl63" style="width:89pt" width="119">11251-35</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">7-0 Prolene suture</td>
			<td class="xl63" style="width:160pt" width="213">Ethicon</td>
			<td class="xl63" style="width:89pt" width="119">W8725</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">6-0 Vicryl suture</td>
			<td class="xl63" style="width:160pt" width="213">Ethicon</td>
			<td class="xl63" style="width:89pt" width="119">W9981</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Cotton buds</td>
			<td class="xl63" style="width:160pt" width="213">Spar</td>
			<td class="xl63" style="width:89pt" width="119"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Millswabs sterile&#160;</td>
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			<td align="right" class="xl63" style="width:89pt" width="119">611950</td>
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			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Drapes</td>
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			<td align="right" class="xl63" style="width:89pt" width="119">141765</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Glucose Saline-Aqupharm3</td>
			<td class="xl67">Animal Care</td>
			<td class="xl63" style="width:89pt" width="119">XVD589</td>
			<td class="xl66"></td>
		</tr>
		<tr height="46" style="height:34.5pt">
			<td class="xl63" height="46" style="width:162pt;height:34.5pt" width="216">Clear H20 HydroGel 98% sterile water&#160;</td>
			<td class="xl67">Clearh2o</td>
			<td class="xl63" style="width:89pt" width="119"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Buprenorphine</td>
			<td class="xl67">National Veterinary Supplies</td>
			<td class="xl63" style="width:89pt" width="119"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Isoflurane vaporizer</td>
			<td class="xl67">General Anaesthetic Services</td>
			<td class="xl63" style="width:89pt" width="119"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:162pt;height:31.5pt" width="216">IsoFlo 100% W/W inhalation vapour, liquid</td>
			<td class="xl63" style="width:160pt" width="213">National Veterinary Supplies</td>
			<td class="xl63" style="width:89pt" width="119"></td>
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			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Sterilizer Steri350</td>
			<td class="xl63" style="width:160pt" width="213">Inotech</td>
			<td class="xl63" style="width:89pt" width="119"></td>
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			<td class="xl63" height="21" style="width:162pt;height:15.75pt" width="216">Microscope Olympus SZX10</td>
			<td class="xl63" style="width:160pt" width="213">Olympus</td>
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			<td class="xl66"></td>
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stenosis of the inferior vena cava: a murine model of deep vein thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

Herein, a model of DVT induced by flow distortion in the IVC is described. Induction of stenosis in the IVC initiates distortion and stagnation of the blood flow and after a while (in this case, 48 h) results in the development of a thrombus, structurally similar to human DVT thrombi (Figure 1A). The presence of a thrombus inside the IVC is easily detectable visually (Figure 1B). An important similarity between the model and human DVT is elevated plasma D-dimer levels (Figure 2). D-dimer is a product of fibrin degradation and its presence in the blood suggests that an active thrombotic process is going on.
The model is presented step-by-step in Figure 3A-C. The IVC is carefully exposed, a hole between the IVC and the aorta is made and a suture (Prolene 7-0) is pulled through the hole beneath the IVC. Then the IVC is ligated by the suture over a spacer (30G needle, Figure 3D), after which the spacer is removed. This procedure allows for about 90% closure of the IVC lumen leaving the remaining 10% patent. This ensures blood flow stagnation eventually leading to thrombosis.
Figure 4 demonstrates the major disadvantage of the model, variable thrombus size. All these thrombi were obtained in the same experiment performed on wild type male mice of the same origin, age and similar body weight. Although all of the mice produced thrombi (thrombosis prevalence of 100%), their size clearly varies in a wide range.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56697/56697fig1.jpg" /> 
Figure 1: Representative thrombus after 48 h restriction/stenosis. (A) a typical thrombus after 48 h IVC stenosis. Note red (red blood cell-enriched) and white (platelet-enriched) parts. (B) IVC with (left) or without (right) a thrombus. Scale bars = 2 mm.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56697/56697fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56697/56697fig2.jpg" /> 
Figure 2: Elevated plasma D-dimer levels after IVC stenosis application. Mice were subjected to IVC stenosis. Blood was taken at the indicated time points, stabilized 1:9 with 3.8% sodium citrate, plasma was prepared by centrifugation (2,300 x g, 5 min) and D-dimer levels were determined by an ELISA kit according to manufacturer&#39;s instructions. Circles designate sham-operated mice, crosses designate IVC stenosis.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56697/56697fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56697/56697fig3.jpg" /> 
Figure 3: Flow restriction/stenosis in the Inferior Vena Cava (IVC) model of DVT in mice. Consecutive stages of the model are presented. (A) a hole between the IVC and the aorta is made and a suture is pulled through it around the IVC. (B) a knot is closed over a spacer (30 G needle). (C) the spacer is removed: the final view that the surgeon sees before closing the mouse. The dashed yellow line demarcates the IVC. LRV stands for left renal vein. Scale bars = 0.2 mm. (D) the spacer (30 G needle). Scale bars =&#160; 2 mm. <a href="https://cloudflare.jove.com/files/ftp_upload/56697/56697fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56697/56697fig4.jpg" /> 
Figure 4. Variability of thrombus size. 
Mice were subjected to 48 h IVC stenosis. Presented are thrombi obtained in the same experiment. Scale bars 2 mm.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56697/56697fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis at JoVE.com

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

stenosis-inferior-vena-cava-murine-model-deep-vein

Research

JoVE Journal

Immunology and Infection

23.7K Views.  University of Birmingham. We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Video: Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

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.

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 ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...