This video demonstrates a streamlined procedure developed to perform metabolic liver studies with sufficient rigor and reproducibility, which can serve as a guide to perform hepatic resection for its vivo perfusion studies. The technique is best applied to real-time metabolism to detect the function, in turnover metabolites in disease models. Perfusion allows precise control of nutrients and hormones available to the liver.
Although the technique is not directed towards therapy, liver perfusions can be instrumental in understanding the etiology and impact of diseases, such as diabetes, NAFLD, and NASH on hepatic metabolism. As new pharmaceuticals are developed, methods for assessing hepatic central energy metabolism become essential. This method can be used with models of several diseases, including cancers and at several different stages of disease progression.
To begin with, place the nose of the mouse into a nose cone after the induction of anesthesia, and taped down its paws. Administer lidocaine through a subcutaneous injection bilaterally in the anterior iliac crest region. Perform a toe pinch test to confirm the absence of all pain reflexes.
Perform celiotomy to expose the internal organs by making a three centimeter wide incision. Expand the incision using a hemostat that pulls the traction by clamping on the xiphoid process. Use a cotton tipped applicator to clear the small and large intestines covering the portal vein.
Position a silk suture under the arch of the portal vein proximal to the liver. Place the second silk suture proximal or distal of the inferior mesenteric vein distal from the liver using a 2-0 suture. Once the sutures are in place, cannulate the portal vein with a 22 gauge catheter, keeping the bevel pointing up.
Enter the portal vein at no more than a 15 degree angle. Tie the first suture past the catheter tapper. After the portal vein is cannulated, anchor the catheter two to three millimeter distal from the branch of the portal vein with the silk suture.
Secure the lower portion of the catheter with the second suture. Tie a knot with the suture to secure the catheter to the distal portion of the portal vein and the surrounding tissue. After the catheter is secured, insert a one milliliter syringe with a 27 gauge needle having 38.1 millimeters length into the catheter to flush blood and air bubbles.
Use a one millimeter inner diameter and five millimeter outer diameter silicone tube with a fixed stop cock to couple the perfusion column to the catheter, allowing the flow of the buffer into the liver, marking the start of the perfusion. After starting a timer, relieve increased vascular pressure by incision using scissors into the inferior vena cava. Confirm flow of perfusate through the liver by observing the homogenous change in liver color from pink or red to a pale yellow.
Once the flow is confirmed, excise the stomach, small intestine, large intestine, and the right kidney from surrounding tissue. With the help of the surgical assistant, maneuver the liver around the abdominal and thoracic cavity as the surgeon cuts through the parietal peritoneum and thoracic tissue to resect the liver. Lift the liver upwards and cut the remaining connective tissues holding the liver in place with scissors.
Slowly manipulate the liver for ease of view. The flow rate and oxygen consumption measurements are vital to monitoring the liver's health and function. There is often a slight difference in oxygen consumption between fed and fasted livers, which can be attributed to increased energy demands imposed by glucogenesis in the fasted liver.
The most important steps include inserting the catheter at a 15 degree angle, visualizing the catheter tip, rolling the wrist and shoulders when tying the suture, and avoiding torsion on the portal vein. The excised tissue can be studied after the perfusion with any number of biochemical or analytical approaches. Ex vivo perfusions in combination with real-time MRI and spectroscopy enable the measurement of metabolic fluxes in real-time.
Naturally, this extends to perturbation of flux from a control state in response to the diseases.
