The overall goal of this procedure is to ligate the IVC causing a complete stasis resulting in a thrombus to study thrombogenesis and resolution. This is accomplished by first exposing the IVC with a ventral midline incision. Next, all back and side draining branches are located and either cauterized or ligated.
Then the IVC is separated from the aorta inferior to the renal veins and ligated. Finally, the IVC and thrombus are harvested at various time points for molecular evaluation. Ultimately, results can be obtained that showed the degree of thrombus burden through day six.
The mouse model of inferior vena cava ligation was developed in our laboratory in order to study gene deleted mice. Visual demonstration of this surgical model is critical as the procedural steps are difficult to learn due to the microsurgical techniques needed to perform this protocol. To begin anesthetize the animal in an anesthetic preoperative induction chamber at 5%isof fluorine, and 100%oxygen at a rate of 0.5 liters per minute.
Remove the animal from the chamber, hold the animal on its back, and shave the ventral abdomen with electric clippers. Next, weigh the animal. Then lubricate the eyes with a sterile ophthalmic ointment.
Place the animal on its back on a warm water heating pad under general anesthesia with isof fluorine and oxygen. Spray the abdomen with chlorhexidine solution and wipe with sterile gauze. Disinfect the abdomen until the surgical site is clean To begin surgery.
Using iris scissors, make ventral midline incision through the skin and abdominal wall, exposing the abdomen using sterile gauze soaked in saline. Deflect the animal's intestines to its left side. Use a mouse restrainer to expose the inferior vanoc CVA or IVC.
From this step forward through the remainder of the surgery, maintain the anesthesia with 2%isof fluorine, 100%oxygen at a rate of 0.2 liters per minute. Using a sterile cotton swab and extra delicate iris tissue forceps. Perform a blunt dissection of the abdominal fascia to expose the IVC and all branches.
First cauterize all the IVC back branches from the renal veins to the iliac bifurcation. Then ligate the side branches with seven oh non-reactive proline sutures. Separate the IVC from the aorta just inferior to the renal veins.
Use seven oh proline sutures to ligate the IVC. Close the abdominal wall using five oh Vicryl synthetic sutures. Then apply adhesive skin glue to close the skin.
Monitor the animal in a recovery cage under heating lamp, then return the animal to its original housing unit. This analysis compares control mice and animals that have undergone IVC ligation and thrombosis at the time of euthanasia. On day two or day six, the IVC thrombus and its associated vein wall were removed and weighed.
The thrombus weight measurement allows the investigator to document the degree of thrombosis and its degree of resolution over time. It is an indicator of the inflammatory event associated with endothelial cell activation. The measurement is also a useful indication of how effective a therapeutic agent is in reducing inflammation and the amount of vein wall thrombus Here, the IVC along with the aorta was removed.
Paraffin embedded sectioned onto slides and stained with hematin and eoin. This graph represents the number of polymorphonuclear cells or PMNs in tissues from control versus experimental mice. The measurement of vein wall inflammatory cell populations is used to document the natural acute to chronic inflammatory response in the vein wall with venous thrombi present in a normal animal, an acute day two inflammatory response should be predominantly neutrophils.
As indicated in this figure, if no infection is present, the vein wall neutrophil population should decrease after day two. In our mouse model of venous thrombosis. Here, the number of vein wall monocytes is compared.
A more subacute and chronic vein wall inflammatory response will show increases. In vein wall monocyte populations monocytes secrete proteolytic enzymes, which have been shown to promote the natural resolution of the venous thrombus. Over time, if infection is not present From this video, you should now have a good understanding of how to perform our IVC ligation model.
This model is great for studying thrombogenesis and resolution of thrombus over time.
