Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis can cause both aortocaval fistula, or AVF, primary failure of maturation, as well as later failure in a working fistula. This technique uses a modified version of an established murine AVF model that recapitulates the clinical course of human AVF and is easily mastered through several critical steps. Before beginning the procedure, confirm a lack of response to toe pinch in a nine-to 11-week-old, C57-Black/6 mouse and place the mouse in the supine position in the surgical area.

Apply ointment to the animal's eyes, and remove the fur from the ventral side of the neck to the lower abdomen. Then cleanse and disinfect the surgical site with three sequential 10%povidone-iodine and 70%isopropanol scrubs, and place a surgical drape over the animal. For clamp and puncture site exposure, place the mouse under a dissecting microscope and, using sterile technique and a scalpel, make a skin-deep, midline, abdominal incision from the lower liver edge to just above the pubis.

Cut through the musculature with scissors to open the abdominal cavity, and insert a retractor into the abdomen. Move the bowels to the right side, and wrap them in a saline-soaked gauze. Move the bladder and the seminal vesicles to the caudal side of the animal, and use a microneedle holder to dissect the mesentery between the rectum and the retroperitoneum to secure a full view of the aorta and inferior vena cava, or IVC.

Then use the microneedle holder to dissect the infrarenal aorta and IVC en bloc from the lateral and dorsal surrounding retroperitoneal tissues to cross-clamp the tissues together, and dissect the surrounding tissues to expose the aortic puncture site at approximately three-quarters of the distance from the left renal vein to the aortic bifurcation. For isolation of the IVC, dissect between the infrarenal IVC and the aorta immediately distal to the left renal vein and extend the dissection distally to halfway between the left renal vein and the aortic bifurcation for post-operative infrarenal IVC observation. Make a window to separate the IVC from the aorta, and dissect the IVC from the surrounding tissue.

Then carefully place an 8-0 polyamide monofilament suture beneath the IVC and the aorta before pulling the suture end through the window to position the suture beneath the IVC only. To create the AVF, first bend a 25-gauge needle to a 45-to 60-degree angle four millimeters from the needle tip and clamp the infrarenal aorta and IVC with a microsurgical clip. Grasping the connective tissue surrounding the bifurcation, rotate the aorta medially and caudally to expose the puncture site of the aorta stretched slightly to the ventral side, and use the 25-gauge needle to puncture through the aorta into the IVC.

When the tissue has been pierced, release the aorta and pull up from the left side of the aorta to cover the puncture site with the surrounding tissue. Then remove the needle, and press the puncture site gently with a cotton-tipped swab for hemostasis. For IVC stenosis creation, place the tip of a 22-gauge intravenous catheter longitudinally onto the IVC and ligate the catheter to the IVC with an 8-0 suture.

When the suture has been placed, remove the catheter and confirm primary hemostasis before unclamping the aorta and the IVC. Cover the puncture site for another minute to ensure hemostasis, and return the organs to their abdominal cavity before closing the abdomen with 6-0 sutures. Then place the mouse into an individual bedding-free cage on a thermal support device with monitoring until full recovery, and use Doppler ultrasound to confirm the patency of the fistula.

On day seven after the surgical procedure, the fistula and stenosis areas of the IVC are easily detected in the longitudinal view. In mice bearing a stenosis without an AVF, the stenosis segment exhibits a venous waveform with more spectral broadening than sham-operated mice but without much pulsatility. In mice having an AVF as well as a stenosis, the stenosis segment shows a pulsatile waveform in addition to spectral broadening, and the time-averaged maximum velocity of the flow at the stenosis in mice having an AVF with stenosis is significantly higher than in mice with stenosis alone.

The mean IVC diameter at the stenosis in mice with stenosis alone is similar to that observed in mice with an AVF and stenosis. In addition, using either the upstream or downstream segment as a reference, the percent stenosis is significantly greater in mice having an AVF in addition to a stenosis. When you ligate the IV catheter and the IVC together, it is important to tie them tightly to create a reproducible stenosis.

The application of this procedure to murine venous graft implantation model could answer how to overcome the occlusion of venous graft stenosis.