Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

The overall goal of this procedure is to modify the murine arteriovenous fistula, or AVF model, into a simple and intergrated method for drug delivery to the fistula endothelium. This method can help answer key questions about the role of the endothelium in venous remodeling. After exposure to the arterial enivroment and in the development of neointimal hyperplasia.

The main advantage of this technique is that it uses a single needle to simultaneously create an AVF while delivering the drug of interest to the endothelium. Before beginning the procedure, autoclave all surgical instruments and vascular clamps. Equip a one-milliliter syringe with a 25 gauge needle and load the syringe with the drug of interest.

Then bend the needle to a 60-degree angle approximately four millimeters from the needle tip and grasp the needle with the curved needle holder. Next, clean the incision site of an eight-week old anesthetized C57 black 6 mouse with a topical antiseptic and place a surgical drape over the animal. Using a scalpel make a mid-line abdominal incision extending from the lower liver edge to just above the pubis and insert a retractor.

Then, transfer the bowels from the abdominal cavity to the right side and wrap them in saline soaked gauze Dissect the membrane connecting the retroperitoneum and lower colon to obtain a full view of the aorta and IVC. To prepare the tissue for the proximal and distal clamp, dissect the infrarenal aorta and IVC from the surronding tissues and place a single microsurgery clip across both the proximal aorta and IVC just below the left renal vein. Place a second microsurgery clip across both the distal aorta and IVC.

Then grasp the connective tissue surrounding the aorta and rotate it medially so that the dorsal surface of the aorta is slightly exposed for the arterial puncture. Keeping the aorta in a rotated position with the left hand, dissect the left lateral margin of the aorta for three quarters of the distance from the left renal vein to the aortic bifurcation so there is ample room to make the puncture with the right hand. Maintaining the aorta in the rotated position, puncture the vessel into the IVC with a curved 25 gauge needle and infuse 100 to 200 microliters of the drug of interest.

The needle will be visible through the dilated thin wall of the IVC as the transparent drug solution displaces the venous blood out of the IVC. Carefully maintain the needle in position for 15 minutes without moving, then remove the distal microsurgery clip from the distal aorta and IVC only. When the distal vessel have been declamped, remove the needle and cover the puncture site of the aorta with the adjacent retroperitoneal tissue.

Remove the proximal microsurgery clip from the proximal aorta and IVC. The arterial blood will be observed flowing into the IVC instead of the usual dark venous blood flow. Take care to provide careful compression when covering the puncture site to avoid occlusion of the AVF and thrombosis.

Finally, after observing 30 seconds of blood flow without compression return the bowels to the abdominal cavity and close the abdomen with a running suture. After closure of the abdomen, apply post-operative care including analgesia, thermal support, and monitoring every 15 minutes until sternal recumbency. In these images, the gene transduction efficiency of this endovascular delivery route with the traditional external route was compared.

As evident, en face observation demonstrated a stronger expression of GFP in the IVC wall in mice treated by intraluminal compared to adventitial delivery of the virus with a characteristic augmentation observed around the fistula at 24 hours post adminstration. Cross sectional histological analysis with anti-GFP antibody showed more robust and site-specific GFP expression along the endothelium of the IVC in mice treated with ILD compared with AD.To confirm these results, infrarenal IVCs treated with adenovirus GFP by either route of delivery were harvested for western blood analysis. Indeed, GFP protein expression was sufficient for detection only after intraluminal delivery, suggesting a superior viral drug delivery to the IVC wall via the intraluminal route at 24 hours post transfection.

Once mastered, this technique can be completed in 25 minutes, including the 15 minute incubation time if it’s performed properly.