Glucose-Stimulated Insulin Secretion via Perfusion through the Mice Vasculature with an Intact Pancreas

We aim to better analyze the function of pancreatic eyelet cells at a level closer to physiological conditions than current research methods. And as an alternative approach for comparison. Our protocol allows for preservation of the pancreas's natural microenvironment to better mimic a physiologic state for further comparison with other techniques For GSIS.

Our laboratory hopes to focus further on implementing the different GSIS methods for the endocrine pancreas, and its response to potential therapeutics like senolytics, which target dysfunctional senescent cells. To begin, identify the upper abdominal aorta by locating the bright red midline vessel with the branching celiac artery, superior mesenteric artery. Ligate superior to the celiac artery and left renal artery.

Then identify the left renal artery. Move the intestines to the animal's left side to gain access to the right kidney. Using forceps, pinch the tissue or fat surrounding the right kidney.

And identify the right renal vein as the dark red or purple vessel connecting the right kidney to the inferior vena cava. Now, identify the abdominal portion of the inferior vena cava as a larger purple vessel with a branch connecting to the right renal vein and perform the ligation. Identify the lower abdominal aorta just before its bifurcation.

Perform the ligation at this point. Then place a suture thread 0.5 centimeters above the first knot. Move the intestines to the animal's left to expose the portal vein.

After making an incision in the diaphragm and ribs, ligate the vessel as superior as possible to ensure space for cannulation later. Place a second suture inferior to the first, but above the pancreas, to ensure collection of the perfusate and avoid backflow. Create a 0.5 to 1.5 millimeter incision using scissors on the lower abdominal aorta between the bottom knot and the superior suture.

Using the lower knot for leverage, insert the tube into the incision via the nick until it reaches the superior suture, being careful not to tear the vessel. Tie the superior suture into a knot to secure the tube in place. Next, create a 0.5 to 1.5 millimeter incision in the portal vein just after it exits the pancreas.

Hold the top knot with blunt forceps in one hand for leverage and insert the tube into the portal vein using forceps in the other hand to guide it in. Tie the second suture to secure the tube. This figure illustrates the insulin secretion dynamics in perfused mouse pancreas in response to varying glucose concentrations and 3-Isobutyl-1-methylxanthine, or IBMX stimulation.

Insulin levels showed a steady low secretion at 2.6 millimolar glucose for the initial 20 minutes, followed by a marked first phase increase upon switching to 16.8 millimolar glucose between 20 and 60 minutes. When the glucose concentration was returned to 2.6 millimolar, insulin secretion sharply declined to baseline levels. Administration of IBMX at 2.6 millimolar glucose caused a strong secondary rise in insulin secretion between 90 and 110 minutes.

The perfused mouse pancreas exhibits a biphasic insulin response to elevated glucose. And retains responsiveness to secretagogues like IBMX, even under low glucose conditions.