Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This method can help answer key questions in the field of advanced liver fibrosis and portal hypertension. It offers a unique opportunity to evaluate pharmacological approaches that target vascular tone, fibrogenesis, or endothelial dysfunction in rats with portal-hypertensive syndrome. The main advantages of this technique include the direct measurement of portal pressure and the opportunity to obtain additional hemodynamic parameters in contrast to the indirect measurement of portal pressure by hepatic venous pressure gradient, HVPG, in patients.

Begin by sedating the rat, using short-time isoflurane anesthesia and optionally additional anesthetics. Quickly proceed to intubation, using a suitable self-made endotracheal tube. Use two fingers to grab the tongue, and gently pull it.

Then, carefully moisten the laryngeal area with a cotton swab soaked in lidocaine spray. Next, gently insert the endotracheal tube between the vocal folds and into the trachea. Fix the endotracheal loop by a transbuccal suture through the cheek and the adhesive tape loop of the tube.

After administering analgesia and injectable anesthesia, apply eye ointment. Then, clip the body hair at the abdominal region and both inner thighs, and disinfect the skin. Assess the anesthesia depth by lid-closure reflex and toe-pinch test prior to any intervention or surgery.

Use tissue forceps to lift the skin on one of the shaved thighs above the presumed location of the femoral artery. Use Mayo scissors to remove a two-centimeter area of skin. Then, use a hemostat to blunt dissect the artery-vein-nerve complex from the connective tissue.

For better visibility of the artery-vein-nerve complex, the cuticle scissor can be used to remove parts of fat tissue. Next, use two high-precision, 45-degree angle, broad-point forceps to separate the femoral artery from the femoral vein and nerve. The femoral vein is the most medial structure, and the femoral artery is located more lateral.

Place a ligature on the femoral artery as distal as possible, and use a curved clamp on the suture to apply gentle longitudinal traction to the femoral artery. Then, place a second, loosely pre-knotted suture on the femoral artery as proximal as possible. Compress the femoral artery at the proximal end of the exposed section, using a micro clamp to temporarily stop the arterial blood flow.

Then, place a supporting micro metal spatula underneath the femoral artery, and use a bent 23-gauge cannula to perforate the anterior arterial wall distal to the occluded section. Carefully catheterize the femoral artery through the perforation with the slanted tip of the catheter facing upward. Advance the catheter until it reaches the micro clamp.

Fix the catheter in its intraluminal location by closing the prepared proximal ligature around the femoral artery and its introduced catheter. Then, open the micro clamp, and check for pulsatile arterial blood flow into the catheter. Fix the catheter position along the vessel, using the ends of the distal ligature to prevent dislocation and to ensure a longitudinal position of the catheter.

Use the digital interface to start recording the heart rate and mean atrial pressure. Begin by performing a median laparotomy. First, lift the skin layer with tissue forceps five to six centimeters below the xiphoid, and use a Mayo scissor to remove a thin strip of skin, using above the linea alba until the xiphoid is reached.

Then, use tissue forceps to lift the muscular layer at the center of the linea alba to create distance between the abdominal wall and the splanchnic organs. Open the peritoneal cavity by incising the abdominal wall with a scalpel at the linea alba. Extend the incision along the linea alba, over the same distance as the skin layer.

Next, carefully excavate the intestine using wet cotton buds. Start with the caecum, and lay it on a large gauze compress, soaked in sterile physiological saline solution placed next to the incision. Wrap the intestine with the gauze compress, and make sure it is moistened with sterile physiological saline solution.

The cirrhotic liver should now be visible. Then, dissect the superior mesenteric artery with two self-made blunt Schwabl hooks, lift the artery with the first hook, and place the second one through the tissue tunnel. Expose the superior mesenteric artery along a five-millimeter distance to ensure the flow probe can be placed around it.

Next, apply ultrasound gel to the ultrasonic flow probe sensor. Attach the sensor to the splanchnic mesenteric artery so that it is aligned to the natural route of the superior mesenteric artery. Measure the superior mesenteric artery blood flow, and assess the accordance of the pulsatile flow signal to the systolic peaks of the femoral artery recording.

To measure portal vein blood flow, first locate and expose the portal vein at the dorsal face of the mesentery that is close to the liver hilum. Then, use high-precision, 45-degree angle, broad-point forceps to gently dissect the portal vein from the surrounding tissue. Enlarge the tissue tunnel, and expose the portal vein along a distance of five to six millimeters to allow the placement of the perivascular flow probe.

Apply ultrasound gel to the ultrasonic flow probe sensor, and attach it to the portal vein aligned with its natural route. Then, close the flow probe, and start recording the portal vein blood flow. To measure portal pressure, wet gloves with physiological saline, and spread the intestine across the fingers.

Optimize the view of the mesenteric vascular bed close to the small intestine. Insert the catheter into the mesenteric tissue by perforating the visceral peritoneum of the mesentery close to the vascular junction chosen for catheterization. Then, carefully advance the slanted tip of the catheter closer to a junction of the ileocolic vein until a slight impression of the vessel junction is seen.

Finally, catheterize the venous system in line with the joining vessel route by perforating the vascular wall at the crossing angle of the vessels. Record all hemodynamic parameters simultaneously under stable conditions for several minutes before euthanizing the animal. The bile duct ligation model causes biliary cirrhosis due to cholestasis.

Accordingly, portal pressure increases over time, and a hyperdynamic circulation develops, as evident by an increased heart rate and decrease of mean arterial pressure. In cirrhotic animals, intrahepatic vascular resistance and superior mesenteric arterial blood flow increase, while subsequently portal venous blow flow rises, depending on the amount of portosystemic collateralization. In contrast, partial portal vein ligation causes prehepatic, non-cirrhotic portal hypertension, which is characterized by an immediate increase in portal pressure.

However, during the time course, portosystemic collaterals develop that may lower portal pressure. In addition, we see corresponding changes in mean arterial pressure and heart rate. For healthy, sham-operated animals, their hemodynamic values remain at physiological levels and do not significantly change over time.

The portal pressure in healthy, SO animals is at maximum five to six millimeters of mercury. Once mastered, these techniques can be performed within 40 to 50 minutes, depending on the animal model, need for troubleshooting, and optimal pre-surgical preparations. While performing the surgical procedure, it's very important to monitor the temperature of the animal, as well as the heart rate and the arterial pressure in order to assess the level of anesthesia.

After watching this video, you should have a good understanding of how to cannulate the femoral artery and the portal vein and how to attach ultrasound flow probes to splanchnic blood vessels in a safe way that will guarantee accurate and reproducible results.