Fluorescence Imaging and Analysis of Blood Flow in Connexin-36 Mouse Pancreatic Islets

Abstract

Pancreatic islets are central in regulating blood glucose levels. Thus, controlling delivery of secreted hormones such as insulin, glucagon, and somatostatin likely depends upon regulating blood flow through each individual islet. The mechanism of control is expected to have one or more components that are localized to the islets, and perhaps to specific cell types within an islet. In wild type mice we have observed localized regulation of blood flow with varying glycemic conditions. Where, under hypoglycemic conditions there is a greater loss of blood flow in islets than in the exocrine pancreas, while for hyperglycemic conditions both islets and exocrine pancreas are well perfused. We hypothesize that this localized regulation of blood flow is related to coordinated electrical activity in islets that couples into vascular endothelial cells to control blood flow. To test this, we use high-speed confocal microscopy to image blood flow in the pancreas of wild type, Cx36(+/-), and Cx36(-/-) (Connexin 36 knockout in beta cells) mice. Data is collected at different z-positions through the islets and surrounding exocrine tissue, for a series of X-Y-Time scans. Vasculature is labeled with a fluorescent rhodamine dextran in the plasma, and a portion of blood cells are labeled by osmotic shock loading with a fluorescent Alexa dye. Results are presented that quantify differences between the three genotypes. Specifically, average blood flow densities and velocities for whole islets and the exocrine pancreas under glycemic conditions from 300 mg/dl (hyperglycemia) are presented. In addition, localized blood flow differences present in single islets, as a function of glycemic conditions, are analyzed. We expect in Cx36(-/-) mice, with islets that lack coordinated electrical activity, much less localized regulation of blood flow under hypoglycemic conditions.