We have established a method for in vivo dynamic imaging of pancreatic islet blood flow and have shown that it can be successfully used to resolve directional patterns of flow in the islet. We believe the novelty of our approach includes not only the in vivo experimental procedure, which incorporates the simultaneous labeling of the β cells (MIP-GFP mice) and the vasculature (rhodamine dextran), but also the real-time 3D imaging capabilities that are essential for capturing the dynamics of blood flow. Prior studies that have investigated patterns of islet blood flow have led to contradictory results, perhaps because they provided only indirect measures of blood flow (16
The 3 putative models of islet blood flow described in previous work have differing consequences regarding how blood flow is coupled to the distinct cellular arrangement of the islet and how it may impact intercellular communication. The 3 models can be described as (a) outer-to-inner, in which blood flows first to the non–β cell perimeter and then to the β cells in the islet core (13
); (b) inner-to-outer, which proposes that the incoming arteries bypass the non–β cell perimeter and first perfuse the β cells (8
); and (c) top-to-bottom, in which the arteries perfuse blood from one side of the islet to the other regardless of cell type (21
). The majority of evidence supporting these 3 models comes from vascular corrosion casting experiments, which are very high resolution but are performed on fixed samples. The few previous live-sample experiments have been complicated by a lack of 3D resolution, making it impossible to discern whether one vessel was in the same vertical plane as another (21
Our results show 2 predominant flow patterns: inner-to-outer and top-to-bottom. The inner-to-outer flow pattern was the most prominent pattern, so it is likely that there are physiological consequences of differing cellular perfusion orders. This pattern supports the concept that insulin or other β cell secretory products can have regulatory effects on downstream cells within the islet (i.e., α cells, which produce glucagon). Consequently, the order in which blood perfuses the islet may be pivotal in the regulation of blood glucose. Because only 1 islet demonstrated an outer-to-inner flow pattern, it is unlikely that secreted products of α cells influence β cells.
The 35% of islets that showed a top-to-bottom flow pattern lends support to the model that proposes that islet blood flow is cell type independent. However, our results do not completely rule out the possibility that islet blood flow patterns are cell type dependent, because we do not know for certain the distribution of non–β cell types within any given islet. It is also possible that irregular structures of islets in vivo, such as nonspherical islets and invaginations, could influence our definitions of what are inner and outer vessels in a way that could make some inner-to-outer flow look more like top-to-bottom. However, it is unlikely that such an explanation would account for all 35% of the islets that exhibited this flow pattern. Finally, because of technical aspects involved in exteriorization of the intact pancreas, our findings are based on the examination of mainly the dorsal region of the pancreas and therefore only depict blood flow patterns of islets in this portion of the pancreas. The present study was also limited to those islets that reside near the pancreatic surface because of imaging depth limitations inherent to confocal microscopy. In the future it may be possible to increase this depth using endoscopic imaging or 2-photon excitation microscopy (29
All prior islet blood flow models describe a large feeding artery that branches into capillaries. Although we did see this type of graduated branching, the larger vessels with numerous smaller vessels extending from them exclusively demonstrated efferent flow. We did find larger vessels with 1 or 2 branches connecting the islet showing afferent flow; however, the diameter of the vessel going into the islet was small and similar to that of islet capillaries. These data represent a comprehensive analysis that we believe was not possible to ascertain in prior studies because of limitations inherent in static in vitro studies.
The functional significance of these findings and why there are 2 predominant, distinct blood flow patterns will require further study. We did not note any recurring pattern of islet architecture, cellular composition, relationship to α islet cells or ducts, or location in a particular region of the pancreas to explain the different patterns of blood flow. The predominance of the inner-to-outer pattern suggests that intercellular communication may be important in the regulation of blood glucose, and therefore replicating the exact milieu of the islet as a cluster of different cell types may be significant for improving transplant studies. Although human islets have a cell type arrangement different from mouse islets (30
), β cells are thought to be grouped together with surrounding non–β cells (6
). Additional work is needed to integrate the current results with mouse islets to human islet blood flow. Understanding the factors that regulate islet blood flow may be important in defining the comprehensive mechanisms involved in islet hormone secretion.
This dynamic study of islet blood flow in time and space has provided information that was to our knowledge previously unattainable and demonstrates how critical in vivo imaging studies with sufficient temporal and spatial resolution are for understanding dynamic biological processes. In addition, data were examined directly and therefore not subject to potential artifacts arising from postprocessing or indirect analysis. Furthermore, these methods can be carried out using reagents and equipment that are now commercially available. The approach described in the present study should be useful for studying the in vivo regulation of pancreatic islet blood flow as well as the dynamics of blood flow in other organs.