In vivo imaging of islet cells and islet revascularization

Abstract: Glucose homeostasis depends on the release of insulin from the pancreatic beta-cell. Impaired insulin release is a hallmark of diabetes mellitus. The beta-cells are situated within the endocrine pancreas, the islets of Langerhans, which are structurally defined microorgans that create a unique microenvironment required for adequate beta-cell function. Pancreatic islet transplantation has emerged as a treatment of type 1 diabetes, but is currently hampered by poor long-term function of transplanted islets. Today, alternatives to monitor islet cell function after transplantation are lacking. Therefore, the aim of this thesis was to develop experimental models that facilitate functional studies of islet cells and islet revascularization after pancreatic islet transplantation under in vivo conditions using fluorescence imaging techniques. Laser-scanning microscopy (LSM) enabled fluorescence imaging in intact islet grafts and functional studies of beta-cells and the islet graft vasculature. LSM was combined with two different transplantation models; ex vivo imaging of islets transplanted under the kidney capsule, and non-invasive in vivo imaging of islets transplanted to the anterior chamber of the eye. To facilitate identification and studies of donor islets after transplantation, the fluorescent reporter expression and function of pancreatic islets were characterized in transgenic YC-3.0 mice. Pancreatic islets in YC-3.0 mice expressed the enhanced yellow fluorescent protein (EYFP), displayed normal beta-cell mass and glucose stimulated insulin release in vitro and in vivo. Furthermore, YC-3.0 islets reversed diabetes and were identified by EYFP fluorescence after transplantation. Islet isolation disrupts vascular connections and thus delivery of oxygen and nutrients to islet cells. Revascularization is therefore vital for the survival and function of transplanted islets. Transgenic Tie2-green fluorescence protein (GFP) mice, characterized by endothelial cell (EC) specific expression of GFP, were used as islet donors. Living ECs were studied in intact Tie2-GFP islets after isolation and during culture. Intraislet ECs survived islet isolation, but rapidly disappeared during islet culture. After transplantation, LSM imaging revealed that donor islet ECs (DIECs) integrated with recipient ECs and formed functional blood vessels during the revascularization of Tie2-GFP islets. Since islet grafts have a deficient vasculature, we investigated if contributing DIECs improved the revascularization of transplanted islets. Freshly isolated and cultured Tie2-GFP islets were therefore transplanted and the contribution of DIECs to the vasculature was determined, as well as the degree of total vascularization and the revascularization rate of the islet grafts. DIECs contributed to the vasculature of fresh but not cultured islet grafts, and fresh islet grafts revascularized faster compared to cultured islet grafts, indicating reduced exposure to hypoxia for fresh islets. However, after completed revascularization the total vascular density was similar in the two groups. Pancreatic islets with beta-cell specific expression of GFP were transplanted to the anterior chamber of the eye. LSM facilitated non-invasive imaging of GFP fluorescent beta-cells in the engrafted islets. Repetitive imaging facilitated longitudinal studies of islet engraftment and revascularization. Furthermore, beta-cell death could be non-invasively monitored in transplanted islets during normal and diabetic conditions. The results in this thesis establish the basis for non-invasive in vivo functional investigations of islet cell physiology and islet revascularization after pancreatic islet transplantation, which can be performed longitudinally under normal and diabetic conditions.