Noninvasive in vivo imaging of pancreatic islet cell biology

Abstract

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laserscanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions. Adequate release of insulin by pancreatic beta cells in response to changing blood glucose levels is a vital requirement for maintaining glucose homeostasis. Failure to do so is one of the major causes of type 2 diabetes mellitus, the most common metabolic disorder in humans 1 . Under physiological conditions, insulin release is regulated by the complex interplay between glucose and a plethora of additional factors-for example, nutrients, autocrine-paracrine signaling and the continuous input from hormones and neurotransmitters 2 . Beta cells, together with other pancreatic endocrine cell types, are situated within the endocrine pancreas, that is, the islets of Langerhans, which are densely vascularized 3 and abundantly innervated 4 . Pancreatic islets, constituting 1%-2% of the pancreatic volume, are difficult to access for in vivo monitoring because they are deeply embedded and scattered in the exocrine tissue of the pancreas 5 . As a consequence, the majority of functional beta cell studies have so far been conducted in vitro on isolated islets or beta cells. Isolated islets 6 , and especially pancreatic slices 7 , allow functional studies of Author Contributions: S.S., D.N. and A.C. developed the experimental transplantation platform. S.S., D.N., O.C., J.Y., R.D.M., A.P., T.M., M.K., B.L. and A.C. did the experiments. J.W. was responsible for generating the transgenic mice. C.R. was involved in designing the transplantation protocols and writing the manuscript. S.S., D.N., I.B.L. and P.-O.B. were responsible for designing the overall experimental plan and writing the manuscript. P.-O.B. was the originator of the idea of using the anterior chamber of the eye for noninvasive in vivo imaging of pancreatic islet cell biology Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions Note: Supplementary information is available on the Nature Medicine website. Laser-scanning microscopy (LSM) of isolated islets and cell preparations has been successfully applied for imaging multiple signaling pathways in the beta cell 6 . However, intravital applications of LSM for studies of beta cell physiology have not been repo...

Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye

“…Techniques used to study the biology of islet of Langerhans in situ, using LSM, were rather invasive and did not allow repetitive imaging at all or only over a limited time period 14,15 . We showed that, after transplantation to the anterior chamber of the eye, isolated islets readily engrafted 13 . Diabetic mice could be rendered normoglycemic by transplanting islets of Langerhans to the anterior chamber of their eye, and these mice showed identical responses to glucose tolerance tests compared to control mice.…”

“…We have recently used the protocol described in this article to transplant isolated pancreatic islets of Langerhans into the anterior chamber of the eye 13 . Studies on vascularized and innervated islets of Langerhans are necessary to understand the mechanisms leading to hormone secretion in health and in diseases such as type 1 and type 2 diabetes.…”

“…We could also repetitively measure systemically induced changes in cytoplas-mic free Ca 2+ concentration in β cells of the same islet. Finally, we noninvasively monitored chemically induced cell death in islets after systemic injection of a β cell toxin 13 .…”

In vivo imaging of murine endocrine islets of Langerhans with extended-focus optical coherence microscopy

“…Essentially, with xfOCM the islets are studied in their natural environment, and disease evolution and islet destiny can be evaluated. This is in contrast to the anterior eye chamber imaging platform [12] that is limited to the study of the engraftment process and graft function in an immunologically privileged engraftment site. Although lacking molecular specificity, xfOCM is potentially able to measure islets in any site accessible to the microscope, provided the islets contrast with the surrounding tissue.…”

“…In recent work [12] islets have been transplanted into the mouse eye. Engrafted on the iris they could be repeatedly imaged using the eye as a natural body window.…”