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

Current methods for glucose-stimulated insulin secretion (GSIS) in rodents involve ex vivo perifusion or static incubation of pancreatic islets and pancreatic slices. Ex vivo perfusion and static incubation allow for multiple samples to be taken quickly with efficiency. However, these procedures subject the islets to trauma, intermittent hypoxia, and reduced paracrine insulin signaling, all of which can affect GSIS. These negative effects can be minimized via perfusion through the mice vasculature with an intact pancreas, which provides another comparison method for the different GSIS models. The backbone of this perfusion procedure involved i) vessel ligation to isolate the pancreas and ii) cannulation of the pancreatic vasculature. This model involved seven ligation points and two cannulations. The resultant perfused vasculature included the lower abdominal aorta, the celiac and superior mesenteric artery, the branching tributaries, and the portal vein. The perfusate was introduced to the vasculature at ~2 mL/min via a peristaltic pump to PE-50 tubing that cannulates the lower abdominal aorta. The perfusates ran through the isolated vasculature and were collected through the portal vein via a cannulation with PE-30 tubing. Perfusate was collected over 3 min intervals in rounds of 30 min for each perfusate solution of 2.6 mM glucose, 16.8 mM glucose, and 2.6 mM glucose with 3-isobutyl-1-methylxanthine (IBMX) in Krebs-Ringer bicarbonate buffer (KRB, 16 mM HEPES and 0.1% BSA, pH 7.4). The IBMX solution was preceded by a normal 2.6 mM glucose solution as a wash cycle. Total time was ~1 h for the surgical procedure and 2 h for perfusate collection. Perfusates were frozen until an ELISA was performed to measure insulin secretion. This method list hopes to provide a detailed procedural basis for insulin secretion measurement through pancreas perfusion through the vasculature, which will allow researchers to perform intact pancreas perfusion successfully in their laboratories.