Microcirculation in Pancreatic FunctionHAROLDWAYLAND*Division of Engineering and Applied Science and Biological Imaging Center, Beckman Institute, California Institute of Technology,Pasadena, California 91125

KEY WORDS pancreas; pancreatic microcirculation; dynamic morphology, transmural trans-port; intravital microscopy, fluorescence microscopy; darkfield illumination.

ABSTRACT The pancreas is involved in two major bodily functions: production of hormonesinvolved in the control of carbohydrate metabolism and the production of enzymes essential todigestion. Pancreatic function is mediated by both neurological and humoral control. The majorpathway for humoral control is through the circulatory system, the level of action being in themicrocirculation. This introductory paper explores the need for a deeper understanding of the dynamicmorphology, i.e. the actual flow patterns in the microcirculation, as a function of the physiologicalstate and demand to complement the careful ultrastructural mapping of the microvasculature. Thecurrent state of knowledge in this field is reviewed as a basis for identifying important areas ofknowledge and ignorance, and some suggestions are made as to possible procedures for furtherexperimental studies, particularly in the microscopic observation of the dynamics of the microcircu-lation with special emphasis on the need for transport studies in both directions across themicrovascular wall.Microsc. Res. Tech. 37:418–433, 1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONThe pancreas is involved in two major bodily func-

tions: the production of hormones important in thecontrol of carbohydrate metabolism and the productionof enzymes essential to digestion. This duality of func-tion was clearly brought to mind by the experience of aphysician friend who, after a severe bout of pancreati-tis, lost the bulk of his pancreatic tissue, but part of itstail was saved. He needed exogenous insulin for a timeafter his operation, but over a period of about 2 years herecovered sufficient hormonal function to have excel-lent control of his carbohydrate metabolism and, forwell over 12 years has had no need for exogenousinsulin, although oral administration of digestive en-zymes is still essential. This would indicate to me thatthe large number of islets in the acinar pancreas isunnecessary for bodily control of carbohydrate metabo-lism, but there is a considerable body of evidence thatthe hormonal production of these islets in the acinartissue is involved in the basic function of that tissue forproduction of the digestive enzymes.It has been well established that there is both

neurological and humoral control of pancreatic functionand amajor pathway for humoral control is through thecirculatory system, the level of action of which is in themicrocirculation. It is the purpose of this paper toexplore our current level of information and ignoranceas to the role of the microcirculation in pancreaticfunction and to suggest possible areas of exploration toalleviate some of our ignorance. Although the majorgoal of this issue is to explore acinar function, the isletsof Langerhans, an important source of the pancreatichormones, and the only source of insulin, are so ubiqui-tous in the pancreas that it seems appropriate toinclude at least some discussion of endocrine functionas well.

The microcirculation is at the locus of most of theimportant interactions between the blood stream andthe surrounding tissue. To understand its role in bodilyfunction there are several aspects of microcirculatorybehavior that we need to try to understand: 1) thedynamic morphology and control of the pancreaticmicrocirculation; 2) the relationship of this flow to theendocrine and exocrine functions of the pancreas; and3) the pathways for themovement of the hormones fromthe endocrine tissue into the blood stream.Broadly speaking, the microcirculatory tree in the

pancreas can be divided into two parts: that which goesdirectly to the parenchymal tissue and that which firstgoes to the islets of Langerhans before either branchinginto the surrounding tissue or draining directly into theportal system. The pancreatic hormones which areproduced within the islets cannot be a part of thefunctional role of the blood which comes directly fromthe blood supplied directly to the acinar tissue. The roleof this part of the microcirculation seems to be less wellunderstood than that which passes through the islets.The complexity of the microvasculature in the acinarlobules is sufficiently great, as shown in the corrosioncasts made by Murakami et al. (1992) in humanpancreata and by Aharinejad et al. (1993) in theexocrine pancreas of the mouse (Fig. 1) that this bloodsupply must certainly be important for the mainte-nance of the tissue itself and probably also having acontrol function in enzyme release through bringingblood-borne signals from other parts of the body, alargely unexplored role. Although the overall architec-ture has been well established, largely by casting

Abbreviations: NO; nitric oxide, L-NAME, Nv-nitro-L-argenine methyl ester.*Correspondence to: Harold Wayland, 900 E. Harrison Avenue, Apt. B-21,

Pomona, CA91767.Received 29 May 1995; revised 20 June 1995; accepted 4 July 1995

MICROSCOPY RESEARCH AND TECHNIQUE 37:418–433 (1997)

r 1997 WILEY-LISS, INC.

techniques, and some of the possible flow patternsestablished by microsphere injection, entirely too littleis known about the ‘‘dynamic morphology’’ of pancreaticmicrocirculation, which can be obtained only by intravi-tal microscopy.An excellent review of themicrovascula-ture (as distinct from the microcirculation) of thepancreas is found in Bonner-Weir (1993).The microcirculation in the islets is both fascinating

and challenging. There are marked species differencesin islet morphology but attention here will be given tothe human islets and those of animal models whichseem to be sufficiently similar to the human to bedirectly useful in improving our understanding of pan-creatic function in our own species. Here the rat, themouse, the hamster and the dog are the animals mostfrequently employed for dynamic, functional studies inthe living animal. To get some perspective on therelationship of the islets to the remainder of the pan-creas we must realize that, according to Bonner-Weir(1993), endocrine tissue represents only 1–2% of thevolume of the adult pancreas but it receives some 10%of the blood volume to that organ. The largest islets,$140 µm in diameter, account for 72% of the isletvolume and 64% of the islet blood flow. The small isletsseem to be largely devoted to supplying hormones to theacinar part of the pancreas, and are distributed through-out this tissue, while the larger islets are more deeplyinvolved with supplying insulin and other hormones tothe body as a whole.

ISLET MORPHOLOGYThe Hormone Producing Cells

The islets of Langerhans contain a remarkable com-plex of hormone producing cells: a cells, which produceglucagon, b cells, which produce insulin, and d cells,which produce somatostatin as well as the PP cellswhich produce a complex of pancreatic polypeptides.The current discussion will be limited primarily to thefirst three types.Aschematic diagram of a ‘‘typical’’ islet

is shown in Figure 2, which also shows the relationbetween the islet and acinar cells and the microvascu-lar structure of the rat pancreas. The interior of suchislets can be thought of as having the a and d cells closeto the outer periphery, or mantle, while the b cells arelargely confined to the core.The microvasculature in the interior of such an islet

is extremely tortuous, as is that in the glomerulus of thekidney. In spite of the great amount of histological workwhich has been done on the morphology of the isletmicrocirculation there is still considerable controversyas to the exact path taken by the blood in the interior ofan islet and to which hormone-releasing cells it isexposed at various stages of its passage.

TheAfferent Blood SupplyLet us first look at what is known about the afferent

arterioles, which will give us some idea of possiblepathways of the blood as it enters the islets, then at theefferent microvessels to try to deduce what type ofhormone load is carried to what part of the pancreas.We will then explore what is known about the dynamicsof these microvascular flow patterns and finally suggestsome experimental challenges to help relieve our igno-rance.The most important issue with respect to the afferent

arterioles is whether or not they pass by the mantlebefore reaching the core or go first to the core and thento the mantle. Morphologically either or both types ofafferent pathways can be found in casts of themicrovas-culature of pancreatic islets, as is beautifully illus-trated in Figure 3 from Murakami et al. (1992) from acorrosion cast of an intralobular islet of a human. Thetwo afferent vessels on the left run first to the mantlebefore entering the core while the one on the right runsdeep into the islet, and this is by no means a uniqueexample. This means that, in some instances at least,the morphology would permit either mantle-core orcore-mantle perfusion or both! It seems unlikely thatboth types of perfusion would take place at the sametime, but this can only be answered by intravitalstudies.

The Efferent MicrovasculatureStudies reported by Nagata et al. (1983) and further

elucidated by Nishino et al. (1985) used a combinationof intravitalmicroscopic, histological and electronmicro-scopic methods to study the microvasculature andmicrocirculation in the pancreas of rats. They proposedessentially three classes of islet microcirculation associ-ated with the efferent vessels, all three of which areshown schematically in Figure 2, although each mightnormally be associated with only one islet.In efferent capillaries of type e-1 the blood flows from

the border of the islet to the exocrine gland in a radialmanner and can serve to evenly perfuse the peri-insular region with islet hormones. In type e-2, one ortwo relatively thick efferent capillaries leave the isletand pursue a straight course toward the tele-insularregion where they anastomose with the exocrine capil-lary network formed by the e-1 type efferent vessels,permitting islet hormones to reach these regions. Thereis some evidence that the acinar cells perfused by the

Fig. 1. Low power electron micrograph of a vascular cast specimendemonstrating the lobular organization of the microcirculation of themouse exocrine pancreas. The capillary loops in the glandular paren-chyma are numerous and homogenously distributed, while regions ofthe interlobular connective tissue are more sparsely vascularized.3110. Courtesy of Dr. S. Aharinejad.

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e-1 efferent vessels are more active in releasing en-zymes than those perfused primarily by the e-2 typevessel. This evidence comes from histological studies inwhich the acinar cells are stained to reveal theirenzymatic activity, showing a distinct difference in thestaining in the two locations (Nishino et al., 1985,personal communication). Type e-3 islets have theshortest of the three types of efferent vessels and emptydirectly into the portal system, so they do not directlyinfluence exocrine function, but provide a rapid trans-port of relatively large volumes of islet hormones to thetarget organs.

Intraislet MicrocirculationThe islet hormones being carried by these efferent

vessels will be determined not only by the possiblepathways of flow in the capillaries within the islets, butalso by the dynamics of that flow. There has beenconsiderable discussion, and frequent disagreement, inthe literature as to the actual pathway(s) taken by theblood in the microvascular plexus in the interior of theislets.

At a Pancreatic Islet Microcirculation Symposiumheld at the Long Beach (California) Veterans Adminis-tration Center on 15April 1994, an international groupof specialists on islet microcirculation discussed threemodels of flow within the islet.Model 1. In this model, which has been particularly

espoused by several Japanese specialists in the field,the flow first passes the mantle before entering the bcell–rich core, so that the blood has the possibility ofpicking up glucagon and somatostatin before it reachesthe insulin producing cells. This would mean that theafferent arterioles would have to enter the mantle sothat the capillary plexus first perfuses the a and d cellsbefore reaching the core with its large population of bcells. Most of the proponents of this as the model haveworked primarily with casts, and hence have no dy-namic data to back up their suggestions. With the wellknown ability of insulin to stimulate the release ofdigestive enzymes from the acinar cells this seems to bea logical manner for islet-acinar interaction, but needsconfirmation by intravital studies.

Fig. 2. Cartoon of an islet of Langerhans schematically showingthe distribution of the a and d cells in the mantle and the b cellsprimarily in the core. The relation between both islet and acinarpancreatic cells and the microvasculature is also illustrated. Inparticular, three types of efferent venules are shown: Type e-1 feeds acapillary plexus near to the islet, type e-2 feeds a capillary plexus somedistance from the islet and type e-3 drains directly into the portalsystem. Reproduced with permission from Nagata, K., Nishino, H.,

Iwasaki, T., Kobayashi, R., Omasa, R., Yoshigoe, F., Hirano, K.,Kuriyama, K., Tamura, T., and Watanabe, Y. (1983) Microcirculatorydynamics and microvascular structure of pancreas in the rat—withspecial reference to endocrine-exocrine relationship for the pancreas.In: Intravital Observation of Organ Microcirculation. M. Tsuchiya, H.Wayland, M. Oda, and I. Okazaki, eds. Excerpta Medica, Amsterdam,pp. 139–157.

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Model 2. In this model, which has been particularlyespoused by Stagner et al. (1988), it is suggested thatthe b cells are perfused before the mantle cells. Theycarried out ingenious experiments on the isolated dogpancreas using antiretrograde (arterial) and retrograde(venous) perfusion of exogenous hormones and antibod-ies to the various hormones. From these studies theyconcluded that, for this particular isolated portion ofthe dog pancreas, the order of perfusion was b = a = d.Albeit this was not an experiment in which the detailedflow dynamics were observed, it did involve a largesample of tissue volume which the more localizedintravital microcirculatory flow experiments are un-

able to do. The same core to mantle flow pattern wasobserved, however, in isografts of golden hamster isletstransplanted into a dorsal chamber in the goldenhamster byMenger et al. (1994). Since hormone produc-tion in the living animal is closely related to functionaldemand, the use even of intravital models in which theislets are placed in an unusual environment needscareful correlation with the actual working environ-ment in which such islets would be found in thefunctioning pancreas.Model 3. In thismodel the blood in the islet microcir-

culation flows from mantle to core to mantle beforeentering the surrounding tissue or exiting into the

Fig. 3. Corrosion cast of an intralobular islet with three afferentvessels (a). The two afferent vessels on the left run from the superficialaspect into the insular capillaries near the periphery of the islet, whilethe one on the right runs deep into the islet. II, interlobular islet; e,efferent vessels (insulo-acinar portal vessels); EL, lobular capillaries.

3450, scale bar 80 µm.With permission fromMurakami, T., Fujita, T.,Taguchi, T., Nonaka, Y., and Orita, K. (1992) The blood vascular bed ofthe human pancreas, with special reference to the insulo-acinar portalsystem. Scanning electron microscopy of corrosion casts. Arch. Histol.Cytol., 55:381–394.

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portal circulation. This pattern was observed dynami-cally using Evans blue dye injection in the rat pancreasby Nishino et al. (1985) and by Liu et al. (1993), also inthe rat, using fluorescent microspheres or fluorescentlylabeled erythrocytes, to follow the path of flow in detailin vivo. A cartoon of the flow pattern visualized by thislatter group of workers can be seen in Figure 4.Critique. It seems reasonable to believe that all of

these types of blood flow within the islets are not onlypossible, but actually take place in the living systemunder functionally appropriate circumstances. In Fig-ure 3, we have seen an islet in a human pancreas whichreceives several afferent vessels, one running deep intothe insular plexus and the others split into its superfi-cial aspect. This would allow the blood to pick upglucagon from the a cells and/or somatostatin from thed cells before reaching the insulin-secreting b cells. Thiswould permit control of insulin release if the pickup ofglucagon and somatostatin were regulated since gluca-gon stimulates the release of insulin and somatostatinhas been shown by Kleinman et al. (1994) to have aninhibitory effect on insulin release. But the vasculararchitecture can also permit core-first perfusion. Herewe need much more critical evaluation of the dynamicmicrocirculatory flow patterns as a function of thephysiological demands on the blood exiting the isletsand an evaluation of just which hormones are beingpicked up by the blood stream under various conditions.Just because amicrovessel passes a particular type of

hormone secreting cell does not mean that it necessar-ily is picking up that hormone. If, as seems almostcertain, some islets, at least, can put out varying mixesof pancreatic hormones, we are led to the necessity of amore detailed study of the mechanisms of control of theislet microcirculation. This will require a clearer under-standing of the nature of the regulatory signals, theirpathways of action, and the actual mechanisms forcontrol of the blood flow and transport of the hormonesinto the blood stream.

MICROCIRCULATORY FLOW CONTROLIn vivo the physiological state of the tissue changes

with functional demand. Unfortunately we know muchtoo little about the particular physiological state of thevarious pancreatic systems which have been studiedeither in vivo or in isolated systems such as the work ofStagner et al. (1988). With what we have learned aboutthe variety of possible microcirculatory flow paths weneed to correlate the actual flow patterns with thephysiologic state of the animal. Here, then, we have acrying need for more dynamic studies, as it is wellknown that the blood flow is not constant in time in allcapillaries. And we need not only to explore the pat-terns of flow, but to try to see if given patterns can beassociated with appropriate regulatory signals.The amount of blood flowing through a given capil-

lary segment will be determined by three things: theeffective viscosity of the blood, the lumen of the vesseland/or the presence of constrictions in the vessel, andthe pressure drop across the segment being studied. Toa first approximation we can content ourselves withconsidering luminal changes as being the most impor-tant.The use of cinematography to make dynamic studies

of microcirculatory flow in the islets is first reported byBunnag et al. (1963). Later McCuskey and Chapman(1969) made dynamic studies of flow in the mousepancreas in which they showed that blood flow in bothacinar and islet capillaries is intermittent, that localblood flow through the acinar capillaries is controlledby smooth muscle precapillary sphincters, while bloodflow in the islets themselves is controlled by a sphincter-like activity of endothelial cells. Although flow in vari-ous capillary segments within an islet could be steady,intermittent, or even reverse, the flow in the feedingarteriole often remains steady within observationallimits.Aharinejad et al. (1993), using dynamic video record-

ing as well as electron micrographic studies, have

Fig. 4. In this islet the supplying arteriole(Art) first delivers blood to capillaries in themantle surrounding the arteriole. The bloodis then carried either to other portions of themantle or to the core of the islet throughnumerous tortuous pathways.Within the core,blood travels to other parts of the core orreturns to the mantle. Blood leaves the isletthrough venules (V) or through islet-acinaranastomoses (IAA). A, D and F (PP) cells arelocated in the mantle while the B cells, whichsecrete insulin, are located in the core. Withpermission from Liu, Y.M., Guth, P.H.,Kaneko, K., Livingston, E.B., and Brunicardi,F.C. (1993) Dynamic in vivo observation of ratislet microcirculation. Pancreas, 8:15–21.

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reported observing endothelial contractions and flowregulation in capillaries of the exocrine pancreas. Itseems likely that many, at least, of the regulatorysignals are blood-borne, involving hormones released inthe islets, and these same signals are also likely to beinvolved in flow control within the islets.The rate of blood flow in the pancreas and to the islets

has been shown by Jansson and Hellerstrom (1993) tobe dependent on the level of blood glucose. The possibil-ity of other blood-borne signals affecting the rate of isletblood flow has not been studied in any detail. It hasbeen well established that hyperglycemia greatly in-creases the flow of blood through islets in the mouse.Recent experiments by Moldovan et al. (1996) haveshown that blocking the production of NO by introduc-tion of L-NAME reverses this hyperemia associatedwith hyperglycemia. The precise locus of the controlwas not observed, although in light of other work bysome members of this same group it seems likely thatthe major control was exerted outside the islet at thepoint where the feeding arteriole branches into amicrocirculatory plexus at the entry to the islet, al-though internal control by endothelial cells cannot beruled out.Liu et al. (1993) determined the dynamic pathway of

perfusion of the capillaries in islets in the rat pancreasby the introduction of a bolus of fluoroscein-labeledserum albumin or by following the paths of fluores-cently labeled erythrocytes. They found that the flow incapillaries in islets in both the head and tail of thepancreas was intermittent, and took tortuous pathwaysof such a nature that there was no consistency as towhich type of endocrine cell was perfused first. If welook at a cartoon of islet circulation as they havevisualized it in vivo (Fig. 4) we see that blood enteringby the arteriole in the upper right divides into severalbranches, two of which skirt the periphery of the isletand are largely involved with a and d cells, while otherbranches go directly into the b cell rich interior. Thedynamics of the flow, rather than the geometry alone,will determine the order of perfusion of the differenttypes of cells.

HORMONAL PATHWAYS FROM ENDOCRINECELLS TO BLOOD

There is a vast accumulation of evidence on themovement of various molecular species from microves-sels into the surrounding tissue, but extremely littlework has been done on movement in the oppositedirection. It is of interest to note that those organswhose function requires the movement of various mo-lecular species into the blood stream, such as theendocrine glands, the peritubular capillaries and theloop of Henle in the kidney, and the villi of the intestine;or a strong capability for such movement, such as thediaphragm, have capillaries with fenestrations closedby diaphragms as shown byMilici et al. (1985) in Figure5. The upper panel is from a pancreatic islet, the secondfrom the intestinal mucosa and the third from a kidneyperitubular capillary. Simionescu (1981), using cation-ized ferritin as a tracer, has shown a strong chargedifferential between the luminal and the abluminalsides of these diaphragms. Figure 6 shows the accumu-lation of such cationized ferritin only on the luminalside of the fenestral diaphragms of an islet capillary,

while Figure 7 is a sketch of the negative chargedistribution on the endothelial wall and in the base-ment membrane of such a capillary. Could such acharge differential play a role in gating the transport inone or the other direction?About a decade ago Elaine Bearer, working in Lelio

Orci’s laboratory in Geneva, was the first to elucidatethe ultrastructure of these fenestrae (Bearer and Orci,1985). Figure 8 shows the ultrastructure of the dia-phragms in the capillary walls of various tissues. PanelA shows the diaphragms in the adrenal cortex, B and Cin the kidney cortex, D in the endocrine pancreas, E inthe exocrine pancreas and F and G in peritubularcapillaries. Such fenestrae have a diameter of the orderof 60 nm. These diaphragms have a very distinctivestructure. They are made up of radial fibrils some 7 nmin diameter, creating wedge-shaped openings with amaximum arc length of 5.46 nm. The central thickeningof these diaphragms had been observed for some time,but the spokelike arms connecting this central core tothe periphery, with gaps between the arms, was onlyelucidated with Bearer’s quick-freeze deep-etch tech-nique. Such diaphragms do not have a lipid bilayer, andappear to contain heparan sulfate proteoglycan (Simio-nescu, 1983).The fact that such diaphragms are quite permeable

to small molecules from the blood stream out has beenpredicted theoretically by Levick and Smaje (1987) andclearly shown by horseradish peroxidase studies. Thesefenestrae should certainly have the capability of allow-ing material to pass either to or from the blood stream.Only recently has convincing evidence been providedthat insulin can pass into the blood stream across sucha diaphragm. This has been shown by Bendayan (1993)using an immunogold technique in which an insulinantibody is attached to colloidal gold particles whichthen can bind with the insulin in a fixed sample oftissue permitting localization of the insulin by means ofelectron microscopy (Fig. 9). The fact that such fenes-trae with diaphragms cover ten times more area in theendocrine capillaries than in the acinar capillaries(Henderson and Moss, 1985) makes them a primecandidate for study of the mechanisms of insulin trans-port into the blood stream. In addition to this differencein the area of fenestrae in the walls of the endocrine andexocrine capillaries, it is interesting to note that thosein the exocrine capillaries appear to be smaller andwith smaller openings than those in the endocrinecapillaries (Fig. 10). A systematic study of the morphol-ogy of the diaphragms in different parts of the pancreashas not been made.Bendayan (1993) has shown that the immunogold-

insulin complex is also found in vesicles, so that vesicu-lar transport may also represent a pathway parallel tothe fenestrae. He used chemical fixation in these experi-ments, and it is well established that the number ofvesicles in chemically fixed endothelial cells can beconsiderably greater than that found in rapidly frozentissue, so the relative importance of these two path-ways cannot be evaluated from histological studies ofchemically fixed tissue. There is evidence that in somesystems, at least, there is a qualitative as well asquantitative difference in vesicular uptake, as shownby Hoying et al. (1995). They studied the uptake ofcolloidal gold-labeled glucosolated albumin by endothe-

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lial cell monolayers (in culture) and found distinctdifferences in the patterns of uptake when the tissuewas cryofixed or chemically fixed. In the chemicallyfixed cells uptake also continued for someminutes afterbeginning of fixation. It is possible, however, to use theimmunogold labeling for the various pancreatic hor-mones on cryofixed tissue, which could reduce theambiguity of such results. Unfortunately, electronmicro-scopic studies are inherently incapable of elucidatingthe dynamics of insulin transport, and light microscopydoes not have high enough resolution to study this

mechanism in detail, so a combination of intravitaloptical microscopy with electron microscopy will beneeded to elucidate the details of many mechanisms.

FUNDAMENTAL PROBLEMS IN STUDYINGPANCREATIC FUNCTION

One of the greatest challenges in trying to under-stand pancreatic function is to evaluate the way inwhich the wonderfully complex islets of Langerhansparticipate in both the endocrine and exocrine needs ofthe body as a dynamical process. There certainly must

Fig. 5. Fenestrae closed by diaphragms (F) in various types ofcapillaries in which an important function is entry of substancesinto the blood stream. C, transendothelial channels; U, unknownstructure. A: Pancreatic islet capillary. B: Intestinal mucosal capil-

lary. C: Kidney peritubular capillary. With permission from Milici,A.J., L’Hernault, N., and Palade, G.E. (1985) Surface densities of dia-phragmed fenestrae and transendothelial channels in differentmurine capillary beds. Circ. Res., 56:709–717.

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be a very finely tuned control system to handle such awide variety of functions.All biological systems must be able to maintain some

semblance of stability in order to function effectively.This characteristic was called homeostasis by Cannonin 1929, a concept which has dominated the study ofphysiological control since that time. Cannon himselfrealized that the term stasis might misleadingly implya complete lack of change, and change is essential forthe operation of all living systems. True stasis issynonymous with death. As we have become increas-ingly aware of the dynamic nature of all living systems,terms such as homeokinesis, or homeodynamics, whichimply dynamic change and stability, seem more mean-ingful. Such dynamic systems must be under constantcontrol to stay within acceptable bounds, and dynamicstudies are essential to help us understand their opera-tion (Fig. 11).Since the various pancreatic hormones are produced

in the islets of Langerhans we must keep in mind thefunctional role of these islets and the fact that theirbehavior in the body may be different from theirbehavior when studied as isolated entities. It hasbecome increasingly clear in recent years that a reduc-

tionist approach, i.e. the study of isolated parts of asystem and an attempt to deduce the behavior of theintegrated system from the properties of these parts,too frequently does not lead to a correct understandingof the operation of biological systems. This is becauseall biological entities are complex systems, made up of adiversity of interacting components whose interactionsare not simple, but non-linear, i.e. the responses ofindividual elements are not simply proportional to themagnitude of the change in the input: doubling theinput does not double the output. Furthermore, theyare also dissipative systems, hence they require energyto function. Figure 12 lists some of the properties ofnon-linear systems which we must keep in mind.In recent years it has become increasingly clear that

complex, non-linear systems may display what is called‘‘deterministic chaos,’’ i.e. they follow deterministiclaws, but may have a region rather than a point ofstability, not returning to a precise control point butstill staying within acceptable bounds. Such systemsfrequently show exquisite sensitivity to small changesin initial conditions, changes which may lead to behav-ior which does not agree with our so-called ‘‘intuition.’’This is the one of the underlying reasons why studies of

Fig. 6. A: Distribution of cationized ferritin on the luminal wall of a mouse pancreatic capillary 2minutes after perfusion. B: Distribution 10 minutes after perfusion. c, transendothelial channel; f,fenestrae; p, plasma lemma; v, vesicle; lumen of the microvessel. 313,600. From Simionescu (1981).

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isolated elements of such a system will not always giveus meaningful clues as to how the system as a wholewill respond to changes in initial conditions. Withrapidly responding dynamic control, however, greatsensitivity to changes in initial conditions can permitspeedy adjustment of response to control signals.We need to keep in mind that all model work needs to

be done in conjunction with studies at the holistic level,whether it be experimental models in which isolatedelements or parts of a system are studied as biologicalentities or whether we are dealing with purely theoreti-cal models. Recent research on the role of ‘‘models’’ inscience (Oreskes et al., 1994) has clearly shown thatcompletely ‘‘correct’’ models can be constructed only forclosed systems, such as found in some branches ofmathematics. All biological systems are open in thesense that we can never know all the possible variablesor changes in their conditions and, in fact, livingsystems continually change either through develop-ment or disintegration. Consequently we can neverexpect to create a ‘‘true’’ model of a biological system.This does not mean that models are useless, usedheuristically they are important, and, I believe, essen-

tial, in guiding us to a better understanding of lifeprocesses. Even our ‘‘intuitive’’ idea as to how a systemmight work is really only a ‘‘model’’ of that system.

SUGGESTIONS FOR FUTURE WORKThis journal issue is dedicated to a better understand-

ing of acinar morphology and function with the goal ofassisting in the design of future research in this impor-tant and fascinating field. Since I feel that the microcir-culation is the ‘‘functional heart’’ of the pancreas, Iwould like to throw out a few challenges which, I hope,may lead to a better understanding of the interrelationof the various entities which make up the pancreas(whether endocrine or exocrine in function) to thehomeokinesis of the islet-pancreas and pancreas-wholebody systems.

Dynamic Morphology Related to FunctionAs has already been discussed, much outstanding

work has been done with corrosion cast techniques toelucidate the microvasculature of both the endocrineand exocrine pancreas, but relatively little in vivo workhas been done to examine actual microcirculatory flow

Fig. 7. Diagrammatic representation of the charge distribution on the capillary wall from the patternof deposition of cationized ferritin. Reproduced with permission from Simionescu, N. (1981) Transcytosisand traffic of membrane in endothelial cells. In: International Cell Biology 1980–81. H.G. Schwinger, ed.Springer, NewYork, pp. 657–672.

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patterns as related to demands made by other parts ofthe body. Although there is probably a good deal ofuseful work which could profitably be done in isolatedpancreata or even with cultured cell types, emphasiswill be placed in this article on direct observation inliving experimental animals. Although some workersseem to feel that the rat, for example, does not have thecapability of core to mantle perfusion in the islets, thework of Liu et al. (1993) and Nagata et al. (1983) andNishino et al. (1985) in Japan have clearly shown invivo that this type of perfusion can and does exist in therat and mouse. It seems almost certain that disagree-ments of this type are related to differences in thefunctional state of the animals during experimentation,and often from overinterpretation of morphologicaldata when not directly related to dynamic intravitalobservations. This puts the need for good dynamic flowstudies related to the functional state high on our list ofexperimental priorities.

Movement of Hormones From the SecretoryCells to the Blood Stream

As we have seen from the pioneering work of Ben-dayan (1993), the fenestrae in the capillaries in theislets are at least one pathway by which insulin canreach the blood within those capillaries. Vesiculartransport is also a possible pathway but the relativerole of these parallel paths has not been established. It

is also important to learn whether or not the other islethormones use either or both of these pathways.

Islet to Acinar-Cell Hormone Transport: Signalsand Mechanisms

We have seen that blood glucose level acts as onesignal for modulating the blood flow within the isletsand is associated with control through the release ofNO. Even for this case the exact mechanisms of controlhave not been ascertained and even less is known aboutthe routes and control of those routes for the passage ofhormones into or out of the blood stream. We need tolearn much more about the nature of both the controlsignals and control mechanisms for these importantbodily functions.

SOME POSSIBLE EXPERIMENTALAPPROACHES

Dynamic Morphology Related to FunctionThe basic methods for studying dynamic microcircu-

latory morphology are essentially the same for anyliving tissue, so the methods suggested will be dis-cussed with respect to islet microcirculation, but wouldbe applicable to the microcirculation in the acinartissue as well. In principle one can design methods forobtaining a great deal of detailed information aboutmicrocirculatory flow and exchange, but in practiceboth time and resource limitations dictate that we

Fig. 8. Fenestral diaphragms in various tissues. A:Adrenal cortex. B, C: Kidney cortex.D: Endocrinepancreas. E: Exocrine pancreas. F, G: Kidney peritubular capillaries. 3361,000. Reproduced withpermission from Bearer, E.L. and Orci, L. (1985) Endothelial fenestral diaphragms: A quick-freeze,deep-etch study. J. Cell Biol., 160:418–428.

427MICROCIRCULATION IN PANCREATIC FUNCTION

carefully design the experimental procedures to eluci-date those features which are of physiological and/ormedical importance rather than merely doing some-thing that hasn’t already been done.Since we are dealing with dynamic systems, under

dynamic control, a major criterion for designing any

experimental procedure will be the time scale on whichthe events of interest take place. In microscopic mea-surements we find that we have something akin to theuncertainty principal in quantum mechanics: one hasto balance the size of the field of view, the magnifica-tion, the spatial resolution and the temporal resolution

Fig. 9. Insulin immunolabeling on a fenestrated area of an isletcapillary. Gold particles are found in the capillary lumen (CL), in thesubendothelial interstitial space (IS) and in the endothelial cell, wherethey are associated with fenestrae (arrows) and plasma membranes

(arrowheads). At (g) we see labeled secretory granules of a b cell.362,500. Reproduced with permission from Bendayan, M. (1993)Pathway of insulin in pancreatic tissue on its release by the B-cell.Am.J. Physiol., 264:G187–G194.

Fig. 10. Structure of the fenestral diaphragms in an endocrine capillary (D) and an exocrine capillary(E). 3637,000. Reproduced with permission from Bearer, E.L. and Orci, L. (1985) Endothelial fenestraldiaphragms: A quick-freeze, deep-etch study. J. Cell Biol., 160:418–428.

428 H. WAYLAND

utilized in any particular experiment. Optimal experi-mental design needs careful focus on the particularphenomena to be studied in each experimental proce-dure. This requires considering a balance betweenspatial and temporal resolution as well as a balanceamong the components used in the observational-recording system. For example, if one is using a videocamera with a CCD chip, the spatial resolution may belimited by the pixel spacing on the chip rather than theoptical resolution of the particular microscope objectivebeing used. And the temporal resolution may well belimited by the integration time required to build up anobservable image on the recording system.Since the basic microvascular patterns have been

well elucidated by means of scanning electron micro-graphic methods, it would seem unprofitable to do invivo studies which merely elucidate these microvascu-lar patterns. Such a method has been nicely demon-strated by Steinhausen et al. (1981) for the glomerulusof the rat kidney. Currently available computer tech-niques would greatly decrease the time and effortrequired for such a reconstruction if a similar task is tobe undertaken. It is much more pertinent, however, toexplore methods capable of effective dynamic resolutionof control of flow, hormone release, and hormone action.Fluorescent tracers are particularly useful for such

dynamic studies. One system which seems to be ofpotential value for pancreatic microcirculatory studiesis the use of acridine red as a fluorescent tracer. Thishas been used by Tangelder et al. (1982) to study themovement of platelets in microvessels. Acridine red can

be used as an intravital dye which stains platelets,leukocytes and, eventually, the endothelial cells. Sinceplatelets and white cells are sufficiently stained to beable to track their motion within the blood vesselsbefore the endothelial cells become sufficiently stainedas to block the observation of the luminal flow, it ispossible to map the flow dynamics and subsequentlyuse the stained endothelial cells to map the morphol-ogy. If, of course, there are luminal changes during thetime in which the endothelial cells are readily observed,these, too, can be studied. Since several minutes ofobservation time are available after injection of theacridine red this would permit control studies of flowand lumen associated with the injection of such sub-stances as glucose or various hormones into the bloodstream as long as the time of action is within theeffective observation time. The peaks of the absorptionand emission spectra of acridine red are 525 and 625nm. Unfortunately the absorption peaks very close to apeak absorption of the hemoglobin in the erythrocytes,which can lead to unwanted heating of the red cells ifhigh intensity illumination is used. On the other hand,the emission peak is far enough in the red that there islittle interference by absorption of that light by the redcells, and the scattering of the light by the tissue andphotochemical effects will be less for the longer wave-length than, say, for the emission of fluoroscein.Platelets stained with acridine red form excellent

endogenous markers for flow studies in the microvascu-lature. As exogenous markers, a wide variety of poly-mer beads stained with various fluorochromes is nowavailable, and the number of fluorochromes available isconstantly increasing.Although red cells can be stainedwith fluoroscein, for example, one has to balance theextremely high quantum efficiency of fluoroscein as afluorochrome against the disadvantages of stimulatingwith light in the 450 to 500 nm range, which can causetissue damage, and the strong absorption of the emittedlight by the hemoglobin in the red cells, which reducesthe visibility of the fluorescent marker.There are significant advantages in using fluoro-

chromes which both absorb and emit at the longerwavelengths. A particularly promising one, and onewhich has been used clinically for studying blood flow inthe fundus of the eye, where fluoroscein angiographyhas been useless, is indocyanine green, better known inthe clinical world as cardio-green. Here the absorptionpeak is around 750 nm, so it can be stimulated with areadily available solid state laser emitting at 780 nmand emits at about 820 nm, which not only reducesscattering by the tissue, but a properly chosen siliconCCD camera is considerably more sensitive in thisregion than in the green, for example.Indocyanine green has two other advantages: it can

be used as an intravital dye since it conjugates sponta-neously to serum albumin (and is virtually non-fluorescent in the non-conjugated state in an aqueoussolution) and it is usable in human beings, having longbeen approved for use in cardiac output and liverfunction studies. Its use in studying the microcircula-tion is reported in Ohshima et al. (1995).If we wish to understand the relationship of the

microcirculation to pancreatic function we really needto be able to work sufficiently deeply into the pancreatictissue to observe microcirculatory flow and behavior

Fig. 11. Properties of biological systems.

Fig. 12. Properties of non-linear systems.

429MICROCIRCULATION IN PANCREATIC FUNCTION

both in islets and in the neighborhood of the acinarducts to be sure we are not dealing with specialproperties of systems near the surface. Here we arefaced with several basic problems: quality of the imagewhich can be formed from an object some hundreds ofmicrons below the surface of the tissue is degraded byabsorption and scattering of the light by the interven-ing tissue; contrast can be seriously degraded due tolight emanating from the tissue layers between theobject and the objective; and it is difficult to supplyadequate illumination of the region to be studied.Confocal microscopy does an excellent job of reducing

the amount of signal coming from the intervening spacebut most confocal systems get full pictures of themicroscopic field only in a matter of seconds. There maybe some events of interest to studies of pancreaticfunction which can be elucidated with data taken thisslowly, but there will be many problems for which thisis completely unsuitable. The tandem scan techniquedeveloped by Petran et al. (1968) using a rotating diskand by Wayland (1989) using an oscillating plate per-mits much fastor recording of individual fields, andcertainly could be used effectively in some situations.The use of ‘‘line scan’’ confocal microscopy also greatlyincreases the speed of image formation. In this ap-proach one has a sort of ‘‘focal plane shutter’’ type ofimage, so that the far edge of the picture is taken laterthan the beginning. This will permit dealing with muchmore rapidly changing dynamic events than point-scanmethods and, by evaluating the effective temporalresolution, there are certainly problems of interestwhich can be studied with this type of system.Fluorescent tracers have many uses besides permit-

ting one to follow microcirculatory blood flow. Fluores-cent antibodies can be used to locate a variety ofsubsystems, such as receptors and hormones, for ex-ample, and tagging different molecular species or anti-bodies with fluorochromes emitting at different wave-lengths can permit studying more than one entityunder the same conditions. With fluorescent tags onemust be aware of the importance of checking whether ornot the tagged species, either through its chemicalproperties or the properties of either the stimulating oremitting radiation, cause unwanted physiological ef-fects. As in all work with fluorescent tracers, it is beingrealized more and more that it is advantageous to havethe emission in the red or near infrared if at all possible.A standard means of stimulating and observing

fluorescent emission is to use the Ploem illuminationand viewing system. In this system (Fig. 13) the sameobjective is used both to illuminate the tissue and toform the image of the area illuminated. The stimulat-ing light and the emitted light are separated by adichroic mirror. This is extremely convenient as onlyone focusing operation is required, particularly if thelens is achromatized for both the stimulating andviewing wavelengths. A very serious drawback to thismethod, however, is that the tissue between the surfaceof the tissue and the plane of observation receives ahigh flux of stimulating radiation, so that any fluoro-chromes in that region will be stimulated and furnish abackground which is often so intense as to wash out thecontrast in the image of interest. This can be consider-ably improved with a form of semi-darkfield illumina-tion, also shown schematically in Figure 13. In this

system the periphery of the lens is used for illuminationand the center for image formation. Even better resultscould be obtained with a darkfield illuminator espe-cially designed for work at some depth within tissue.Unfortunately, the only epi-darkfield illuminators nowavailable have been designed for work on the surfacerather than below the surface of the object to bestudied. The efficiency of utilization of the light sourcefor darkfield illumination can also be greatly increasedover the standard approach. Since a hollow cylinder of

Fig. 13. ‘‘Ploem’’ illumination for stimulation of fluorochromes byepi-illumination. Both the stimulating light and the fluorescent emis-sion are focused by the same objective. In the standard ‘‘Ploem’’ systemthe stimulating light fills the entire cylinder which then enters thefield from a dichroic mirror which reflects the shorter wavelengths buttransmits the fluorescent emission. This system effectively eliminatesrecording the scattered stimulating light but, if there are fluoro-chromes between the surface of the tissue and the desired plane ofobservation, this can lead to significant loss of contrast in the desiredimage. The optical path in this case is marked by broadly spaced slantlines. By confining the stimulating beam to a hollow cylinder whichonly illuminates the periphery of the objective and forming thefluorescent image with the center of the objective, a form of semi-darkfield stimulation is possible which can considerably increase thecontrast in fluorescent microscopy at depth in tissue. The optical pathin this case is shown by the finely hatched areas.

430 H. WAYLAND

light is needed to illuminate such a condenser, it hasbeen customary to expand the beam from the lightsource into a solid cylinder and then vignette out thecenter of the beam (Fig. 14A). By the use of an axiconilluminator (Fig. 14B) it is possible to concentrate muchmore of the light into the hollow cylinder. This methodis particularly useful when using laser illumination asit is relatively easy to obtain a suitable cylindrical beamof parallel light of the desired diameter from such asource. For any observations at depth within biologicaltissue the objective should be designed to permit correc-tion for the distance the light travels in the tissue. Inthe case of a water immersion objective the correction issmall enough that it can often be neglected as the indexof refraction of the tissue and that of water will bereasonably close to each other. If, however, there is anair interface between the objective and the tissue therewill be optical errors introduced by the relative distancethe light must travel in the two differing media. Correc-tions similar to those made for cover glass thicknessbecome desirable if the lens has a numerical aperture

greater than about 0.2, and become quite significantwith apertures above 0.3. At present there are a veryfew objectives made for use in inverted microscopes inwhich corrections can be made for Petri dish thick-nesses up to 2 mm. When used as dry objectives thiswould permit corrections for even greater depths withintissue.There are no commercially produced darkfield con-

densers which are particularly effective for illumina-tion below the surface of biological tissue. We areundertaking a design study of such illuminators at theBeckman Institute at the California Institute of Technol-ogy, but it will be some time before we have prototypesready to study with tissue.Shibata et al. (1995) have recently published an

ingenious method in which two ribbonlike beams froman Argon laser are so focused as to cross at the desireddepth below the tissue surface (Fig. 15) and used tostimulate the fluorescence of fluoroscein compounds inthe blood stream in capillaries. The field which isuniformly illuminated is about 50–60 µm 3 1 mm and

Fig. 14. A: Conventional method of obtaining a hollow cylinder oflight for darkfield illumination.Aparallel cylinder of light is formed bythe condenser lens and the center is blocked out by a stop to form ahollow cylinder of light. B: Axicon illuminator. A narrow cylindrical

beam of parallel light is converted into a disk of light by a conical 45°mirror. This disk is then converted into a hollow cylinder of light bymeans of a 45° mirror on the surface of a truncated cone.

431MICROCIRCULATION IN PANCREATIC FUNCTION

about 30 µm deep. If the long axis is made parallel tothe axis of a capillary, this permits observation of boththe capillary lumen and sufficient extravascular spaceto make near-field diffusion studies from the capillarywall. This system could certainly be modified to permitscanning, either of a complete microscopic field or of amore limited area so that the temporal resolution couldbe increased in such a smaller region.

Movement of Hormones Into and Out ofMicrovessels

The brilliant work of Bendayan (1993) in demonstrat-ing the possible role of the fenestrae as a pathway forthe entry of insulin into the blood stream needs furtherelaboration. Certainly work using the same type ofimmunogold techniques could be carried out with cryo-fixed tissue which could help us determine the relative

importance of fenestrae and vesicles in insulin take-upby the blood stream, and the use of appropriate antibod-ies to somatostatin and glucagon could also be used tostudy their pathways of entering the blood stream.Unfortunately such electron microscope methods in-volve fixing the tissue so that in general it is oneanimal–one experiment for one particular set of circum-stances. There are, however, some questions which areimportant enough to justify this tedious type of work.For example, we know that a high glucose load in theblood significantly increases the rate of blood flow inislets. Does this result in an increase in the uptake ofinsulin by the blood and what is the actual pathway forentering the blood stream? It would seemworthwhile toutilize the immunogold technique to answer this ques-tion and possibly similar questions with respect to theother islet hormones.Some interesting intravital studies can also be con-

ceived to answer important questions relating to hor-mone take-up and transport. Insulin can be tagged witha fluoroscein compound and, hopefully, with a fluoro-chrome which emits in the red or near infrared toreduce scattering and avoid the absorption of thefluorescent emission by hemoglobin. By introducingexogenous insulin which has been tagged with a fluoro-chrome into an islet, preferably by microiontophoresisrather than by micropuncture, one could, hopefully,measure the fluorescent pickup in a draining venule asa function of glucose level of the blood perfusingthat islet. Local introduction of the blood at variousglucose levels, or blockage of the pathways involvedin neural control of insulin secretion, could also beilluminating. If there is a positive correlation be-tween the ease of passage of the insulin into thebloodstream and the level of glucose in the blood at thelocal level, then it would be interesting to see if thecharge on the diaphragms has been changed withdifferences in glucose concentration. Possibly this couldbe done using cationized ferritin as a label on thecharged surfaces. If, indeed, there is a significantchange in the negative charge, this would be strong,although not completely definitive, evidence thatmodu-lation of the charge on the diaphragms was involved inthe control function of insulin movement into the bloodstream.The above examples are given only to show the type

of experimentation which is now possible, experimenta-tion which needs the collaboration of multidisciplinaryteams versed in pancreatic function, intravital micros-copy, electron microscopy, biochemistry, etc.

IN CONCLUSIONAn amazing amount has been learned about the

structure and ultrastructure of pancreatic microcircula-tory beds by means of histological methods, enhancedwith the use of polymeric casts viewed with the electronmicroscope. Unfortunately, all of these methods giveonly an indication of the morphological arrangement ofthe fixed tissue, which is often distorted by the fixationprocess. From such data there is no way that one candeduce with any certainty just what the path of theblood stream is in the living tissue. We must eventuallyappeal to a systematic study of what I have calleddynamic morphology. A good case in point is found inthe studies of the microcirculation of the islets which

Fig. 15. Schematic of method of using two slitlike beams of laserlight to stimulate fluorescence at depth within the tissue withminimalstimulation in the intervening space between the surface and the locusof observation. A: A three-dimensional view showing the relationshipof the laser beams andmicroscope objective to the tissue.B:Asectionalview showing how the light from the laser beams does not stimulatethe fluorescence except near the focal region of the microscopeobjective. Reproduced with permission from Shibata, M., Kawamura,T., Sohirad, M., and Kamiiya, A. (1995) A new fluorescence microscopyfor tomographic observation of microcirculation by using dual-beamslit laser illumination. Microvasc. Res., 49:300–314.

432 H. WAYLAND

have been described above. The paucity of solid informa-tion is clearly brought out by the fact that there arestrong partisans for each of the models which weredescribed earlier in this paper, and which I havesuggested are all correct for some one ormore physiologi-cal states. Eventually we must carefully consider avariety of possibly disturbing circumstances such as theeffect of the particular anesthetic used, the operativeprocedures, and the metabolic state of the animalduring experiments. (It is pretty common practice to doanimal surgery on fasting animals. How relevant is thisto studies of pancreatic function?) And another factorwhich is important in all biological models is the size ofthe sample observed. It is here that an interactionbetween theoretical and experimental models can oftenbe of great help in limiting the amount of observationalwork that needs to be done.Another area which needs further elucidation is the

relationship between the dynamicmicrocirculatory pat-terns in the islets and acinar tissue and their functionin the homeokinesis of the animal. There is evidencethat the majority of islets which are primarily associ-ated with an endocrine function in the other parts of thebody are larger than those peppering the acinar tissue.One needs to establish if the microcirculatory patternsare intrinsically different in the ‘‘large’’ and ‘‘small’’islets, or whether there are primarily temporal differ-ences which can best be understood by the nature of thecurrent demands on the blood from those islets.There is also evidence that there is oscillation in the

rate of release of insulin. Does the oscillatory behaviorof insulin secretion occur in all normal states? Does itshow deterministic chaos in its behavior? There arecases in biological systems in which chaotic behavior isshown in the rest state but oscillation in a stimulated,operational state. And we can find any correlationbetween blood flow patterns in themicrovessels and thesecretory patterns from the various endocrine andacinar cells?I cheerfully admit that I lean strongly toward the

need for critical studies at the level of intravital micros-copy, and feel that we now have many powerful toolswhich have not been adequately exploited. But bythemselves, such studies would be primarily a form ofmental masturbation. To be fruitful they need to becarried on in collaboration with studies from a widevariety of disciplines and constantly tested against thelatest knowledge of pancreatic function.

REFERENCESAharinejad, S., MacDonald, I.C., Schmidt, E.F., Bock, P., Hagen, D.,and Groom,A.C. (1993) Scanning and transmission electron micros-copy and high resolution intravital video-microscopy of capillaries inthe mouse exocrine pancreas with special emphasis on endothelialcells. Anat. Rec., 237:163–177.

Bearer, E.L. and Orci, L. (1985) Endothelial fenestral diaphragms: Aquick-freeze, deep-etch study. J. Cell Biol., 160:418–428.

Bendayan, M. (1993) Pathway of insulin in pancreatic tissue on itsrelease by the B-cell. Am. J. Physiol., 264:G187–G194.

Bonner-Weir, S. (1993) The microvasculature of the pancreas, withemphasis on that of the islets of Langerhans. Anatomy and func-tional implications. In: The Pancreas: Pathobiology and Disease,2nd ed. V.L.W. Go, E.P. Dimagno, J.D. Gardner, E. Lebenthal, H.A.Reber, G.A. Scheele, eds. Raven, NewYork, pp. 759–768.

Bunnag, S.C., Bunnag, S., and Warner, N.E. (1963) Microscopy of theliving pancreas in situ. Am. J. Anat., 126:395–408.

Cannon, W.S. (1929) Organization for physiological homeostasis.Physiol. Rev., 9:399–431.

Henderson, J.R. and Moss, M.C. (1985) A morphometric study of theendocrine and exocrine capillaries of the pancreas. Q.J. Exp. Physiol.,70:347–356.

Hoying, J.B., Chen, S.C., and Williams, S.K. (1995) Interaction ofcolloidal gold-labeled glucosolated albumin with endothelial cellmonolayers: Comparison between cryofixation and glutaraldehydefixation. Microsc. Res. Tech., 30:252–257.

Jansson, I. and Hellerstrom, C. (1993) Stimulation by glucose of theblood flow to the pancreatic islets of the rat. Diabetologia, 25:45–50.

Kleinman, R., Ohning, G., Wong, H., Watt, P., Walsh, J., and Brunica-rdi, F.C. (1994) Regulatory role of intraislet somatostatin on insulinsecretion in the isolated perfused human pancreas. Pancreas,8:172–178.

Levick, J.R. and Smaje, L.H. (1987) An analysis of the permeability ofa fenestra. Microvasc. Res., 33:233–256.

Liu, Y.M., Guth, P.H., Kaneko, K., Livingston, E.B., and Brunicardi,F.C. (1993) Dynamic in vivo observation of rat islet microcirculation.Pancreas, 8:15–21.

McCuskey, R.S. and Chapman, T.M. (1969) Microscopy of the livingpancreas in situ. Am. J. Anat., 126:395–408.

Menger, M.D., Vojkowsky, P., Berger, C., and Messmer, K. (1994)Orientation of microvascular blood flow in pancreatic islet isografts.J. Clin. Invest., 93:2280–2285.

Milici, A.J., L’Hernault, N., and Palade, G.E. (1985) Surface densitiesof diaphragmed fenestrae and transendothelial channels in differ-ent murine capillary beds. Circ. Res., 56:709–717.

Moldovan, S., Livingston, E., Zhang, R.S., Kleinman, P., and Bruni-cardi, F.C. (1996) Glucose induced islet hyperemia is mediated bynitric oxide. Am. J. Surg., 171:16–20.

Murakami, T., Fujita, T., Taguchi, T., Nonaka, Y., and Orita, K. (1992)The blood vascular bed of the human pancreas, with specialreference to the insulo-acinar portal system. Scanning electronmicroscopy of corrosion casts. Arch. Histol. Cytol., 55:381–394.

Nagata, K., Nishino, H., Iwasaki, T., Kobayashi, R., Omasa, R.,Yoshigoe, F., Hirano, K., Kuriyama, K., Tamura, T., and Watanabe,Y. (1983) Microcirculatory dynamics and microvascular structure ofpancreas in the rat—with special reference to endocrine-exocrinerelationship for the pancreas. In: Intravital Observation of OrganMicrocirculation. M. Tsuchiya, H. Wayland, M. Oda, and I. Okazaki,eds. Excerpta Medica, Amsterdam, pp. 139–157.

Nishino, H., Ozawa, K., Takeishi, M., Nagata, K., and Watanabe, Y.(1985) Pancreatic microcirculation in rats (particularly the islet ofLangerhans). In: Microcirculation Annual 1985. M. Tsuchiya, M.Asano, M. Oda, and I. Okazaki, eds. Excerpta Medica, Amsterdam,pp. 325–337.

Ohshima, N., Homma, S., Yanagi, K., Sato, M., Ito, T., andWayland, H.(1996) Intravital near-infrared fluorescencemicroscopywith indocya-nine green to visualize deep tissue microcirculation. In: TissuePerfusion and Organ Function. T. Kamada, T. Shiga, and R.S.McCuskey, eds. Elsevier, Amsterdam. pp. 15–27.

Oreskes, N., Shrader-Frachette, K., and Belitz, K. (1994) Verification,validation, and confirmation of numerical models in the earthsciences. Science, 261:641–646.

Petran, M., Hdravsky, M., Egger, M.D., and Galambos, R. (1968)Tandem-scanning reflected-light microscope. JOSA, 58:661–664.

Shibata, M., Kawamura, T., Sohirad, M., and Kamiiya,A. (1995)Anewfluorescencemicroscopy for tomographic observation ofmicrocircula-tion by using dual-beam slit laser illumination. Microvasc. Res.,49:300–314.

Simionescu, N. (1981) Transcytosis and traffic ofmembrane in endothe-lial cells. In: International Cell Biology 1980–81. H.G. Schwinger,ed. Springer, NewYork, pp. 657–672.

Simionescu, N. (1983) Cellular aspects of transcapillary exchange.Physiol. Rev., 63:1536–1579.

Stagner, J.I., Samols, S., and Bonner-Weir, S. (1988) b = a = dpancreatic islet cellular perfusion in dogs. Diabetes, 37:1715–1721.

Steinhausen, M., Zimmerhackl, B., Thederan, H., Dussel, R., Parekh,N., Esslinger, H.U., von Hagen, G., Komitowski, D., and Dallenbach,F.D. (1981) Intraglomerular microcirculation. Measurements ofsingle glomerular loop flow in rats. Kidney Int., 20:230–239.

Tangelder, G.J., Slaaf, D.W., and Reneman, R.S. (1982) Fluorescentlabeling of blood platelets in vivo. Thromb. Res., 28:803–820.

Wayland, J.H. (1989) Scanning Microscopy. U.S. patent number4,806,004.

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