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.

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.