Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

The overall goal of this unique rat liver hilar clamp model is to enable the study of the impact of pharmacologic molecules of interest in ameliorating ischemia reperfusion injury. This method can help answer key questions in the field of liver transplantation about which molecules may be directly infused into the liver to help mediate ischemia reperfusion injury. The main advantage of this technique is that the molecule of interest is directly injected into the portal vein, reducing the distribution volume.

The implications of this technique extend toward the transplantation of all organs, as it allows the testing of molecules that might be added to organ perfusates for ischemia reperfusion injury reduction. Visual demonstration of this method is critical because of the small size of the portal vein branch used for cannulation. Begin by confirming the appropriate level of sedation by toe pinch, then place the rat on a warming pad under a dissecting microscope and restrain the limbs.

Next, make a midline abdominal skin incision from the pubis to the xiphoid, followed by a similar incision in the peritoneum along the linea alba. Enter the abdomen, taking care not to damage the bladder or bowel, and release the liver from the peritoneum anteriorly near the xiphoid process. Make a transverse incision through the skin and the peritoneum at the inferior border of the right lobe of the liver, and use a curved mosquito clamp to retract the xiphoid process.

Use rib retractors to open the ribs as far as possible from the midline, and cut the falciform, phrenic and gastric ligaments. Then use moistened sterile cotton swabs to flip the liver up and cut additional ligaments as necessary to gain access to the porta. Using saline moistened gauze, viscerally rotate the liver, then remove the loose connective tissue overlying the portal hilum, and the loose connective tissue overlying the length of the portal vein.

Using forceps, push through the loose connective tissue posterior to the left portal vein, artery and bile duct to make a window and loosely encircle the vessels with a 4-0 Potts suture. Then clear the loose connective tissue overlying the posterior branch to the portal vein at the right kidney. Next, use an insulin syringe to draw 0.5 milliliters of blood from the inferior vena cava and transfer the blood into a small vial.

Centrifuge the sample and collect the serum in a new vial for snap freezing in liquid nitrogen. Cut two pieces of 7-0 suture. Place the first 7-0 loop around the left portal vein as far medial as possible, and use a curved mosquito clamp to retract the vein.

Place a loosely tied second 7-0 loop on the posterior branch to the portal vein near its intersection with the portal vein. Now, load the infusion pump with a five milliliter syringe containing three milliliters of reagent in 0.9%saline, and prime the tubing. Use a microsurgical clamp to grip the distal portal vein to reduce bleeding during the cannulation, and use small microsurgical scissors to cut a 0.5 millimeter hole in the vein between the 7-0 stay suture and its intersection with the portal vein.

Using a 27-0 catheter, cannulate the left portal venous system, inserting the catheter past the bifurcation of the left and right portal veins. Infuse one milliliter of normal saline to confirm the placement of the cannula and watch for the left lateral and median lobes of the liver to blanch. Manually assess that the catheter is past the takeoff of the right portal vein, but not beyond the takeoff of the portal vein feeding the median lobe, then cinch the Potts suture, secure the catheter with the 7-0 suture, and remove the clamp from the distal portal vein.

Immediately begin the infusion of two milliliters of the reagent of interest over a 15 minute delivery period, with continuous monitoring of the animal's vital signs throughout the infusion. One hour from the beginning of the ischemic time, remove the Potts suture and the catheter, then cinch the 7-0 suture on the posterior branch to the portal vein. Two hours after beginning the reperfusion, slowly draw 0.5 milliliters of blood from the inferior vena cava, followed by slow drip transfer of the blood into a new vial.

Apply gentle pressure to stop the bleeding from the vein as necessary. Then centrifuge the blood and snap freeze half of the serum for downstream processing, as just demonstrated. At the experimental endpoint, incise the diaphragm in a circle and sever any additional connective tissue that remains connecting the liver to the peritoneal cavity.

Collect the liver and obtain four large samples from the left and median lobes, and four large samples from the right lobe. Then place the samples into individual labeled vials and snap freeze the tissues in liquid nitrogen for downstream processing. Serum alanine aminotransferase measurement in normal saline and PEG-SOD infused hilar clamped animals demonstrates a significant difference between the serum control and experimental animal liver enzyme expression levels at 120 minutes post-reperfusion.

Tissue malondialdehyde expression in the right, non-hilar clamp lobe does not exhibit a significant difference between the control and experimental groups. The left post-hilar clamp and reperfusion lobe, however, demonstrates a significantly higher level of tissue malondialdehyde expression compared to the control non-reperfused, non-infused lobe, as well as to the right PEG-SOD infused lobe. The same pattern is also observed for tissue glutathione expression.

Western blot analysis indicates an increased level of cleaved caspase-3 expression in the left lobe after hilar clamp and reperfusion that is decreased in animals treated by PEG-SOD infusion, differences which are confirmed to be significant by densitometry. Once mastered, this technique can be completed in three hours and 30 minutes if it is performed properly. While attempting this procedure, it is important to remember to obtain substantial traction on the portal vein branch for cannulation when placing the distal portal vein clamp.

Once this procedure is mastered, it can be used to explore the impact of a variety of pharmacologic substances. After watching this video, you should have a good understanding of how to cannulate the left portal venous system.