194-OR: A Versatile Platform for Intravital Imaging of Biosensor-Labelled Beta Cells In Situ

Abstract

The pancreatic islet is a complex system containing numerous cell types, including endocrine, immune, and endothelial. The pathologies of T1D and T2D originate in this organized, multicellular environment in vivo, and thus can only be fully understood in this context. We have developed several adaptable tools to generate a versatile platform combining intravital microscopy (IVM), virally-packaged biosensors, and abdominal imaging windows (AIW) allowing us to gather β cell specific measurements in vivo longitudinally. Here, we present characterization of in vivo physiology of mouse β-cells using two virally-packaged biosensors roGFP2 and GcAMP6s. To achieve in vivo expression of the biosensors, we used IP injected AAV8 to transduce in situ β cells. Using redox-sensitive roGPF2, we measured the response in biosensor signal after IV administration of the cytotoxic glucose analog alloxan from islets in situ (∼5-fold change vs. saline). To test the portability of the platform, we utilized Alox15 knockout mice (Alox15-/-), which show protection against oxidative stress. We observed a rapid 4.6-fold increase in biosensor signal ratio in WT mice, compared to a 3.3-fold increase in Alox15-/- mice after IV alloxan treatment. Using AAV8-GcAMP6s, we measured calcium activity of β-cells in situ after a glucose bolus and detected a small sub-population of β cells that oscillate at a 2x frequency compared to the entire islet. To overcome the endpoint nature of standard IVM, we next implanted AIWs over the pancreas of biosensor-labeled mice, which allows for stable, longitudinal imaging of islets in situ over for several weeks. Using IVM through AIW’s in AAV8-roGFP2 transduced mice, we monitored individual islets, before, during, and after streptozotocin-induced β cell ablation. We measured a ∼75% reduction in islet area by 10 days post ablation. Altogether, this versatile platform can lead to novel experiments examining β-cell physiology and signaling in vivo longitudinally in a variety of unique scenarios. Disclosure C.A. Reissaus: None. S.A. Tersey: None. K.W. Dunn: None. A.K. Linnemann: None. R. Mirmira: None.