Assessing Insulin Clearance in Mice via In Situ Liver Perfusion

Insulin clearance plays a key role in regulating glucose metabolism, and is linked to metabolic disorders such as obesity, insulin resistance, and the diabetes matters. The accurate assessment of insulin clearance is crucial. This protocol outlines a user-friendly hepatic perfusion procedure in mind for directly evaluating hepatic insulin clearance rate.

Insulin clearance is estimated in humans, and animal models using both direct and indirect methods. However, few studies have employed liver perfusion to access hepatic insulin clearance directly in a mouse model. The liver perfusion technique maintains the liver underneath the larger conditions by circulating a warm oxygenated, and a nutrient enriched proficient through the liver vascular.

The success of surgical catheterization is crucial for the liver profusion system due to the slander blood vessels in mass. Accurate placement of catheters in the portal vein and inferior vein cover is particularly critical. If the catheter is inserted too deeply, it may cause uneven perfusion between the liver lobes, which can affect the test results. The mouse liver perfusion system is valuable tool for investigating the dynamics and the molecular mechanism of hepatic insulin metabolism. In addition, this protocol could be widely used not only in pre induced disease models, but also in acute challenge stimulus testing.

[Narrator] To begin, place the anesthetized mouse on the operation table and secure the limbs using adhesive tape, make a four centimeter longitudinal incision from the lower abdomen toward the xiphoid process along the mid abdominal line. Using scissors, carefully cut the peritoneum to avoid damaging the visceral organs. Insert the abdominal retractor to expose the surgical field. Move the intestines to the right to reveal the portal vein, right kidney and inferior vena cava. Using artery forceps, clamp the vena cava at the upper edge of the kidney. Next, isolate the portal vein, and ligate the distal end with a 6-0 Silk suture. Loosely tie another suture on the proximal end of the exposed vessel. Make an incision near the ligated end with spring scissors and insert the catheter. Advance the catheter through the incision up to the level of the portal bifurcation. Now, secure both ligatures around the catheter, and confirm proper sampling by connecting the free end of the catheter to a sampling syringe. Then flush the heparinized saline, and clamp the catheter. Next, remove the traction device, and reset the intestines. Cover the surgical area with saline-soaked sterile gauze or cotton. Make an incision along the sternum from the xiphoid process exposing the sternum. Then vertically cut open the sternum, and cut through the diaphragm along the rib margin to expose the thoracic cavity. Expose and isolate the super hepatic inferior vena cava. Then carefully ligate the distal end with a 6-0 Silk suture. Loosely tie another suture on the proximal end of the vessel. Next, make an incision just below the ligated end with spring scissors. Insert a 10 centimeter catheter through the incision and advance it until the tip of the catheter is close to the liver. Then tie both ligatures securely around the catheter. Confirm proper sampling and clamp the free end of the catheter. Finally, rinse the surgical area with saline, and cover the surface with saline soaked sterile gauze. To begin, set up the liver perfusion system, which includes an oxygenator, a temperature modulation device, an infusion pump and infusion tube. Turn on the water bath, and prewarm the organ chamber to 37 degrees Celsius. Prime the tubing system with the perfusion buffer incubated in the water bath. Place the euthanized mouse in a container with a warming pad to maintain a temperature of 37 degrees Celsius. Then connect the liver perfusion system to the portal vein catheter. Use a mini pump to infuse the KRBH buffer through the portal vein catheter at a controlled rate of 0.2 milliliters per minute. Observe the liver turning pale within seconds indicating perfusion buffer flow. To flush out remaining blood cells in the liver, pause the infusion for one minute at the four minute, and eight minute time points starting the timing at the beginning of the perfusion. After 10 minutes, collect the basal sample from the inferior vena cava catheter. Next, perfuse the liver with the KRBH buffer enriched with four nanograms per milliliter of human insulin for an additional 30 minutes. Collect the samples from the inferior vena cava tube every two minutes. At the end of perfusion, record the liver weight, and collect liver samples from different lobes. Centrifuge all collected perfusion samples at 1,000 G4 five minutes at four degrees Celsius. Collect the supernatants, and transfer them to minus 80 degrees Celsius for storage. High fat diet feeding led to a reduced hepatic insulin clearance rate with approximately 25% clearance compared to 55% in the control diet group. Liver tissue structure appeared normal in well perfused samples with neatly arranged hepatic cords. Intact hepatocytes and clear nuclei. Poorly perfused liver samples showed emus hepatocytes, vascular degeneration, nuclear hypnosis, and punctate necrosis around central vein. Levels of alanine aminotransferase, and aspartate aminotransferase showed no significant difference between baseline and post perfusion measurements.