In vivo imaging of islet cells and islet revascularization
Year: 2007
Type: dissertation
Abstract: Glucose homeostasis depends on the release of insulin from the pancreatic\nbeta-cell. Impaired insulin release is a hallmark of diabetes mellitus.\nThe beta-cells are situated within the endocrine pancreas, the islets of\nLangerhans, which are structurally defined microorgans that create a\nunique microenvironment required for adequate beta-cell function.\nPancreatic islet transplantation has emerged as a treatment of type 1\ndiabetes, but is currently hampered by poor long-term function of\ntransplanted islets. Today, alternatives to monitor islet cell function\nafter transplantation are lacking. Therefore, the aim of this thesis was\nto develop experimental models that facilitate functional studies of\nislet cells and islet revascularization after pancreatic islet\ntransplantation under in vivo conditions using fluorescence imaging\ntechniques. Laser-scanning\nmicroscopy (LSM) enabled fluorescence imaging in intact islet grafts and\nfunctional studies of beta-cells and the islet graft vasculature. LSM was\ncombined with two different transplantation models; ex vivo imaging of\nislets transplanted under the kidney capsule, and non-invasive in vivo\nimaging of islets transplanted to the anterior chamber of the eye.\n\nTo facilitate identification and studies of donor islets after\ntransplantation, the fluorescent reporter expression and function of\npancreatic islets were characterized in transgenic YC-3.0 mice.\nPancreatic islets in YC-3.0 mice expressed the enhanced yellow\nfluorescent protein (EYFP), displayed normal beta-cell mass and glucose\nstimulated insulin release in vitro and in vivo. Furthermore, YC-3.0\nislets reversed diabetes and were identified by EYFP fluorescence after\ntransplantation.\n\nIslet isolation disrupts vascular connections and thus delivery of oxygen\nand nutrients to islet cells. Revascularization is therefore vital for\nthe survival and function of transplanted islets. Transgenic Tie2-green\nfluorescence protein (GFP) mice, characterized by endothelial cell (EC)\nspecific expression of GFP, were used as islet donors. Living ECs were\nstudied in intact Tie2-GFP islets after isolation and during culture.\nIntraislet ECs survived islet isolation, but rapidly disappeared during\nislet culture. After transplantation, LSM imaging revealed that donor\nislet ECs (DIECs) integrated with recipient ECs and formed functional\nblood vessels during the revascularization of Tie2-GFP islets. Since\nislet grafts have a deficient vasculature, we investigated if\ncontributing DIECs improved the revascularization of transplanted islets.\nFreshly isolated and cultured Tie2-GFP islets were therefore transplanted\nand the contribution of DIECs to the vasculature was determined, as well\nas the degree of total vascularization and the revascularization rate of\nthe islet grafts. DIECs contributed to the vasculature of fresh but not\ncultured islet grafts, and fresh islet grafts revascularized faster\ncompared to cultured islet grafts, indicating reduced exposure to hypoxia\nfor fresh islets. However, after completed revascularization the total\nvascular density was similar in the two groups.\n\nPancreatic islets with beta-cell specific expression of GFP were\ntransplanted to the anterior chamber of the eye. LSM facilitated\nnon-invasive imaging of GFP fluorescent beta-cells in the engrafted\nislets. Repetitive imaging facilitated longitudinal studies of islet\nengraftment and revascularization. Furthermore, beta-cell death could be\nnon-invasively monitored in transplanted islets during normal and\ndiabetic conditions. The results in this thesis establish the basis for\nnon-invasive in vivo functional investigations of islet cell physiology\nand islet revascularization after pancreatic islet transplantation, which\ncan be performed longitudinally under normal and diabetic conditions.
Source: Figshare
Author: Daniel Nyqvist
Language: English
Subfield: Endocrinology, Diabetes and Metabolism
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1771572070