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 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.
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'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'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.
All procedures were approved by the institutional Animal Care Committee of McGill University Montréal, QC, Canada. All the equipment required is listed in Table I.
1. Murine (C57BL/6J) IVC Stasis Protocol
2. High Frequency Ultrasound Protocol
NOTE: This protocol is adapted from the Lady Davis Institute Rodent Phenotyping Core SOP for high frequency ultrasound imaging. This protocol is carried out 24 h post-operative, but can be done sooner as long as the animal is responding well to the surgery. The protocol can be carried out at any time on healthy mice and is often done so to compare before and after surgery.
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 branches are ligated with a 6-0 silk suture (Figure 2). Then, the IVC is separated from the aorta by blunt dissection and the silk is placed around the IVC (Figure 3A-C). The IVC is ligated with a 6-0 silk suture. Dilatation of the IVC below the ligation site is an indication of successful interruption of the blood flow (Figure 3D). Finally, the peritoneal cavity and the skin are sutured back in a continuous manner (Figure 3E).
Monitoring of thrombus formation using ultrasonography
As we have previously shown, high frequency ultrasound, which is commonly used to assess venous thrombosis in clinical settings, can be used to measure thrombus formation and resolution over time in an experimental murine model. We used a high frequency micro-imaging system12. Prior to ligation, the IVC can be identified in the longitudinal view. After ligation of the vein, the success of the procedure can be visualized. The PW-Doppler mode can be used to determine blood flow velocity before (34.8 mm/s) and after (5.6 mm/s) the ligation. Because thrombi are denser than flowing blood, we could appreciate the formed thrombus inside the IVC using ultrasonography, 24 hours after the ligation (Figure 4A). Ultrasonography allows for quantification of the velocity of blood flow in the vessels using color Doppler. As shown in Figure 4B, we can measure the flow in the vein prior to ligation and appreciate the interruption of the flow after the ligation, when the thrombus is formed. Data from our laboratory show an average thrombus size of 4.85 ± 0.22 mm2 at 24 hours and 5.05 ± 0.47 mm2 at 48 hours (mean ± SEM).
Figure 1. Stasis model: Procedure to expose the inferior vena cava. (A) Mouse hair from the abdomen is removed using hair removal cream. (B) A first incision is made on the left side of the abdomen and a second ventral one (C) from left to right. (D) A wet sterile gaze is used to exteriorize the intestines and expose the inferior vena cava (IVC) and side branch (SB). Please click here to view a larger version of this figure.
Figure 2. Stasis model: Procedure to ligate the side branch. (A) Exposition of the IVC and SB. (B) Placement of the suture silk underneath the SB. (C) Ligation of the SB. LRV: left renal vein. Please click here to view a larger version of this figure.
Figure 3. Stasis model: Procedure to ligate the inferior vena cava. (A-B) Blunt dissection to separate the IVC from the aorta. (C) Placement of the suture silk underneath the IVC. (D) Ligation of the IVC. Dilatation of the IVC is observed. (E) The abdominal muscles and skin are closed separately. Please click here to view a larger version of this figure.
Figure 4. Monitoring of thrombus formation using ultrasound imaging. (A) Representative ultrasound images before, immediately after and 24 hours after ligation of the IVC. (B) Representatives images of blood flow velocity depicted by color Doppler using color processing. The scale of color-coded blood flow ranges from red to yellow to depict high to low flow. In the ligated animals the absence of flow is depicted by the absence of color. Please click here to view a larger version of this figure.
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 °C incubator for at least 30 minutes after the surgery and to return it to the company of other animal only after full recovery. When the surgery is done correctly, the animals behave remarkably well during post-operative care. They exhibit no serious side effects such as lameness, paresis, or incontinence. They may exhibit reduced movement and a slightly hunched posture immediately after surgery, but this is not seen often as long as the analgesic is effective and the surgery performed properly.
The stasis model produces a large thrombus with reproducible size measurements from one mouse to another. As in humans, the anatomy of the venous system varies between mice and the issue of IVC branch interruption has recently been addressed in the stenosis and the stasis models of DVT13,14. Brandt et al. showed that thrombus formation induced by flow restriction was prevented when the side branches were located at <1.5 mm of the IVC ligation site. However, DVT formation induced by flow restriction in mice was not affected by side branch ligation13,15,16. It was also reported that the variability of IVC side branches in C57Bl6 mouse strain has an important impact on thrombus formation induced by complete ligation of the IVC14. It was found that not ligating side branches results in statistically smaller clot size compared to controls with ligated side branches. Our study also suggest that ligation of the side branches produced consistent thrombus formation and size. However, the most frequent anatomical variation in C57Bl/6 is the presence of 2 back branches (98% of the mice)14. It was demonstrated that interrupting back branches has the biggest impact on thrombus size. The present methodology did not address the effect of back branches, which can be interrupted using a low temperature cautery pen14. However, we demonstrated that ligation of the IVC and side branches resulted in consistent thrombus formation in C57Bl/6. Finally, as we have previously demonstrated, the high frequency ultrasound system allows the precise measurement of thrombus size and can be used for long-term and translational studies of thrombus formation and resolution12,13,15.
One major disadvantage of the stasis model is the complete obstruction of blood flow, which reduces the maximal effect of intravenously administered agents on the thrombus and vein wall. This becomes an important issue when one wants to test the effect of a pharmacological agent. If the effect of one specific drug needs to be tested, one would prefer using the stenosis model13 or the electrolytic model17. Both models produce thrombi in the presence of continuous blood flow, which allow testing of new anti-coagulant agents for DVT prophylaxis and treatment.
The authors have nothing to disclose.
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.
6-0 perma-hand silk suture | Ethicon | 706G | |
Surgical Scissors | Fine Science Tools | 20830-00 | |
Suture tying forceps | Fine Science Tools | 20830-00 | |
blunt forceps (straight and curved) | Fine Science Tools | 20830-00 | |
Needle Driver | Fine Science Tools | 13002-10 | |
Moria Spring Scissors | Fine Science Tools | 15396-00 | |
1ml syringes | BD Biosciences | ||
26G needles | Becton Dickinson & Co. | ||
VEVO 770 High Resolution Imaging System | Visualsonics | No longer sold | |
SR Buprenorphine | ZooPharm | Given to LDI by Vet | |
Surgery Microscope | Leica | Leica M651 | |
Systan eye oinment | Alcon | 288/28062-0 | |
2×2 sterile Gauze | CDMV | #104148 | |
Cotton Tip Applicators | from JGH | ||
Transpore hypoallergenic surgical tape | CDMV | #7411 | |
Ultrasound gel (Aquasonic-100) | Dufort & Lavigne | #AKEN4061 | |
Incubator | From JGH | ||
Isoflurane | Dispomed | ||
Anesthetic chamber,hoses, and adminstration equipment | Dispomed | ||
Hair remover | Nair | ||
Water heated hard pad | Braintree Scientific, Inc. | #HHP-2 | |
Gaymar heater water pump TP500 | MATVET Inc. | #R-500305 | |
Infra-red heating lamp | electrimat inc. | #1R175R-PAR | |
Mouse rectal temperature prope | emkaTECHNOLOGIES | ||
Sterile water | From JGH |