Microcirculation in the Islets of Langerhans of the Mouse' SRICHITRA C. BUNNAG: SIROTMA BUNNAG A N D NANCY E. WARNER Department of Pathology, The University of Chicago, Chicago, Illinois A technique for the study of microcirculation in living islets, using ABSTRACT quartz rod and cinephotomicrography, is presented, together with results of a study of insular microcirculation. With this technique, conditions are standard, results are reproducible and the findings are recorded permanently. The pancreas, bathed constantly in Locke-Ringer's solution at 37"C, is lifted with the spleen from the body, supported on the tip of Knisely's hollow-tipped fused quartz rod, and the lobules are separated gently. Circulation i n the transilluminated islets, which are pale yellow, spherical or ovoid bodies with a distinctive vascular pattern, is studied with the biobjective, binocular microscope. For cinephotomicrography, the camera is aligned over one eyepiece of the microscope and the film is exposed at 64 frames per second, using a 750 watt projection bulb as light source. Direct anastomoses of insular and acinar capillaries are not observed. Blood flow i n the islets of the anesthetized mouse is rapid and constant, under these conditions of observation. Intravenous epinephrine and ephedrine cause temporary interruption of blood flow in the islets, whereas norepinephrine slows but does not stop the circulation. Pitressin also causes slowing of insular blood flow. Insulin, glucagon, hydrocortisone, glucose, alloxan and diphenylthiocarbazone have no acute effect upon the insular microcirculation. A possible relation between the circulatory effect and the hyperglycemic effect of epinephrine and ephedrine is suggested. Few studies of microcirculation in the islets of Langerhans in living mammals have been reported, and observers do not agree concerning normal patterns of blood flow (Kuhne and Lea, 1882; OLeary, '30; Berg, '30a, b; Brunfeldt, Hunhammar and Skouby, '58; Palmer, '59). That the results reported are contradictory appears to be due to the widely varied experimental conditions, and the difficulty in recording, and hence interpreting, the transitory phenomena of microcirculation. Using the quartz rod for transilluminatior, and cinephotomicrography, we have developed a technique for the study of microcirculation in living islets. With this method, standard experimental conditions are obtained, results can be reproduced and there is a permanent record of the findings, permitting objective analysis by more than one observer." MATERIALS ANT) METHODS Animals The animals used were male and female Webster Swiss and AKr white mice, C,H brown mice and Holtzman white rats. In most of the experiments, Webster Swiss mice were used. The mice weighed between 20 and 30 gm. The diet was Purina Laboratory Chow and water, ad libitum. The anesthetic used was sodium pentobarbital (Nembutal Sodium, Abbott). The initial dose was 1 m g per 10 gm of body weight, administered by intraperitoneal injection. Narcosis was achieved within 10 minutes, and was maintained by repeating the injection every 45 to 60 minutes. The tail veins were used for intravenous injections. Anatomy of the pancreas The gross and microscopic anatomy of the pancreas was studied by conventional dissection and histologic sections, supplemented by intravital injection of 0.2 ml of diphenylthiocarbazone solution, 4 ml of 1 Supported by grants from the United States Public Health Service (H-3944) and the Louis G. Block Fund, The University of Chlcago. 2 Submitted i n partial fulfillment of the requirements for the degree of Master of Science, Department of Pathology, The University of Chicago. 3 Preliminary findings in this study were published as an abstract, Anat. Rec., 139: 337, 1961. A 16 mm color motion picture which documents part of this work was shown at the Ninth Microcirculatory Conference and at the Annual Meetlng of the American Association of Anatomists, March, 1961. A copy of this film is available for loan. 117 118 SRICHITRA C . B U N N A G , SIROTMA BUNNAG A N D N A N C Y E. W A R N E R undiluted Higgin's India ink, or both. Diphenylthiocarbazone, a chelating agent which combines with zinc in the islets to form a visible red complex, was prepared according to the method of Brunfeldt, Hunhammar and Skouby ('58). h e - h u n dred-twenty mg of diphenYlthiocarbazone were dissolved in 1.5 ml of 95% ethanol, two drops of NH4OH were d d e d and the mixture was diluted to 20 ml with distilled water. For intravenous injection, 0.2 ml of the final solution was used. Gross dissection of the Pancreas was done in 20 mice. In Six of these mice7 India ink was injected into the left ventricle prior to dissection. Microscopic examination of the whole, fresh pancreas after intravital injection was done in ten mice. Prior to injection, collateral observation of the normal, intact miCrOCirCUlatiOn Was made. In four of these mice, the inferior vena cava was ligated above the diaphragm and 4 ml of undiluted India ink were injected into the vena cava below the diaphragm. In three mice, the inferior vena cava was ligated above the diaphragm and the injection of ink was followed by injection of 0.2 ml of diphenylthiocarbazone. In three mice, the injection of ink followed by diphenylthiocarbazone was made into the left ventricle. The islets were measured and counted in ten mice. Six Of these mice were Six weeks old, and four of the mice were three months old. The mice were sacrificed by exsanguination, and 0.5 ml of diphenylthiocarbazone was injected rapidly and forcefully into the splenic pulp. The pancreas was then completely excised, placed whole upon a Zeiss slide micrometer, and examined with a binocular, monobjective microscope, using reflected light, a 3.5 X objective and 10 X oculars. Study of t h e islets in vivo The microcirculation in the pancreas was studied in 300 living mice, by the following method. A 1 cm left abdominal incision was made in the anesthetized mouse, and the inferior pole of the spleen, with attached pancreas, was lifted gently from the body. The exposed tissues were bathed constantly with Locke - Ringer's solution, at 37°C. The pancreas was transilluminated by hollow-tipped, fused quartz rod, supporting the organ on the tip of the rod without compression, and the lobules were teased apart gently, avoiding hemorrhage. The apparatus used for transillumination and irrigation of the tissue was devised by Knisely ('54). To find islets, the pancreas was examined minutely, first with the unaided eye, and then with dissecting microscope. The Leitz biobjective, binocular microscope, equipped with 12.5 x oculars and 2 x, 4 x , 8 x and 12 X objectives was used. With this microscope, perception of depth was possible, and superimposed structures could be studied. In reflected light, the islets were tiny, discrete, spherical or ovoid white bodies, situated along the blood vessels and ducts. When transilluminated, they were brilliant and pale yellow, with a distinctive vascular plexus, which contrasted with the paler, less vascular parenchyma. The larger the islet, the more intense was the color. If hemorrhage occurred during the search for islets, the animal was discarded. Criteria of normal circulation outlined by Knisely, Warner and Harding ('60) were followed, and animals in which abnormalities were recognized at the outset were not used. Diphenylthiocarbazone (0.2 ml) was injected intravenously at the end of most experiments, to confirm that structures observed were islets. Frozen sections were made in some instances, but this cumbersome procedure was abandoned as proficiency in identifying islets in vivo was attained. Cinephotomicrography The microcirculation in the islets was photographed with the Eastman CineKodak Special I1 camera, using Kodachrome Type A film. The camera was modified by adding a motor-drive, and the ground glass in the viewfinder was replaced with clear glass. When photographing through the microscope, no lens was used on the camera. Using the biobjective microscope, the field to be photographed was selected. The camera, supported by tripod, was aligned directly over one of the eyepieces of the microscope, and viewing of the microscopic field and critical focussing were done through the camera's viewfinder thereafter. The cam- 119 MICROCIRCULATION IN ISLETS era and eyepiece were connected by a lighttight tube of black cotton fabric. The shutter-lever of the camera was left in the open position. The light source was a 750 watt projection-lamp bulb, at 120 volts. The film was exposed at 64 frames per second, and projected for viewing at 16 or 24 frames per second. 0.2 ml was injected. Hydrocortisone was supplied in a solution containing 50 mg per ml. Dilution to 0.2 m g was made, and 0.2 ml, containing 0.04 m g of hydrocortisone, was injected. Normal saline was used for dilutions, except in the case of diphenylthiocarbazone, which was prepared as described above and used undiluted. Drugs and chemicals The substance to be tested was administered by intravenous injection or by local application dropwise to the islet, under direct observation. Each substance was tested in ten or more mice. The microcirculation was studied first in each mouse, to be certain that no abnormality existed. The substances used were : epinephrine, norepinephrine, ephedrine, pitressin, insulin, glucagon (supplied by Eli Lilly and Company), alloxan, glucose, hydrocortisone and diphenylthiocarbazone. Epinephrine was prepared from a 1: 1,000 solution, 1 ml of which contained 1 mg of epinephrine. Dilutions of 1:10,000; 1:20,000; 1 : 100,000, 1 :500,000, 1:750,000; and 1: 1,000,000, in volumes ranging from 0.01 to 0.03 ml were used. Norepinephrine was prepared from a solution containing 1 mg of 1-norepinephrine per ml. Dilutions containing final doses of 0.4, 0.8 and 2.4 Ug in 0.1 ml volumes were used for intravenous injections. For local application, solutions containing 4, 8, 24 and 1,000 ug of 1-norepinephrine per ml were used (0.0004%, 0.0008%, 0.0024% and 0.1% , respectively). Ephedrine was supplied in a solution containing 50 mg per ml; 0.05 ml of the solution, containing 2.5 mg of ephedrine, was injected. Pitressin was prepared from a solution containing 20 pressor units in 1 ml. Dilutions of 1:400; 1: 1,000; 1: 10,000; and 1:200,000 were made, and volumes of 0.01 to 0.03 ml were used. Regular insulin (Squibb U40, USP) was administered undiluted in doses of two units, five units and ten units, in volumes of 0.05 ml, 0.12 ml and 0.25 ml, respectively. Glucagon was supplied in a solution containing 1 mg per ml, and 0.2 ml, containing 0.2 mg, was injected. Alloxan was prepared in 3% solution, and 0.2 ml, a n amount containing 1 mg per 5 mg of body weight, was injected. Glucose was obtained in 10% solution, and Histological techniques Tissues were fixed in Bouin's solution or in 10% formalin. Paraffin sections were cut at 3 or 4 p, and stained with hematoxylin-eosin, Gomori's chrome alum hematoxylin phloxine stain (Gomori, '41), and Cason's modification of the Mallory Heidenhain stain (Gurr, '56). RESULTS Anatomy of the pancreas The pancreas in the mouse and rat is dendritic, with long, thin, branching lobes in a clear membranous mesentery. The pancreas of the mouse is easier to transilluminate because it is smaller and thinner. In the mouse, as in the rat (Greene, '35), the arterial supply is derived from the celiac and the superior mesenteric arteries, and the venous blood is drained by the portal vein. The lobes of the pancreas, termed tertiary lobules by Opie ('03), are composed of secondary lobules, each with a n artery, vein and branch of the pancreatic duct. The secondary lobules, in turn, are composed of primary lobules, each with a n arteriole, venule and ductule. In the fresh, whole pancreas after injection of diphenylthiocarbazone, the islets are stained bright red and are easy to identify. Selective staining occurs whether injection is intravital or postmortem. The maximum diameter of the islets measured ranges from 10 to 500 p. The largest islets, which are usually ovoid or beanshaped, are found close to the largest vessels. The total number of islets in the mice six weeks of age ranges from 123 to 183; i n the mice three months of age, the number ranges from 134 to 219. With double injection of India ink and diphenylthiocarbazone into the left ventricle, the vessels in the acinar parenchyma and the islets are clearly demonstrated, and the islets are stained red as well. In- 120 SRICHITRA C . B U N N A G , SIROTMA B U N N A G A N D N A N C Y E. W A R N E R jection of ink into the ligated vena cava readily fills the acinar veins, capillaries and arteries, but does not fill the vessels of the islets. Double injection of ink and diphenylthiocarbazone into the cava does not fill the islets consistently, and staining is not uniform. Histologic study of the islets in untreated animals confirms the grouping of beta cells in a central nidus, with a mantle of alpha cells at the periphery. Blood s u p p l y of the islets These observations are based upon the study of the living pancreas, coupled with study of the injected specimens. Arterioles can be differentiated from venules easily in the live animal, by means of the arteriolar pulsations, the bright red color of arteriolar blood, and the direction of flow from larger to smaller channels. Each islet has one or more afferent arterioles, and the larger islets have two or three (fig. 1). When two or more arterioles are present, they enter the islet at the same or opposite sides. Most islets derive their arterial supply from lobular arteries, via arterioles which may supply a branch to the acinar parenchyma before reaching the islet. Some islets are closely apposed to the pancreatic ducts (fig. 2 > , and these islets may be supplied by arterioles which arise from mural vessels of the ducts themselves. A few islets are located close to the splenic artery and are supplied by short arterioles which arise directly from that artery. Fig, 1 Islet of Langerhans in the living mouse. Afferent arterioles (A) at upper left, and efferent venules ( E ) a t lower left, of islet. Photomicrographic enlargement of individual frame of 16 mm. Kodachrome motion picture. Original magnification 150 X. Fig. 2 Islet of Langerhans, with afferent arteriole ( A ) , efferent venule ( E ) , and branch of pancreatic duct (D). Photomicrographic enlargement of individual frame of 16 mm. Kodachrome motion picture. Original magnification 150 X , The arterioles divide just inside the islets, to form a rich network of capillaries. Direct anastomoses of insular capillaries and the adjacent capillaries of the acinar parenchyma have not been observed. The capillaries drain into short venules, one to six in number, depending on the size of the islet. These venules form at the boundaries of the islets, or just beyond. These venules merge and enter the lobular veins, which ultimately drain into the portal vein. Microcirculation in the islets The flow of blood in the pancreas of living, anesthetized mice is extremely rapid, under the conditions of these observations. Individual erythrocytes cannot be distinguished, even at the highest magnification used. The flow of blood in the capillaries is constant, and spontaneous interruption of flow, action of sphincters, “leukocyte-blocking’’ (Palmer, ’59) and reversal of flow are not seen. The pancreas can be observed continuously for three hours or more before signs of circulatory abnormality begin to appear. Most often, the first change is adherence of leukocytes to the lining of vessels, and intravascular agglutination usually appears next. Epinephrine injected intravenously in solutions of 1:1,000 to 1:20,000 has a marked effect on the circulation in the islets. The arterioles and venules are constricted, and there is temporary interruption of the flow of blood in the capillaries. The arterial pulse rate is increased. The 121 MICROCIRCULATION I N I S L E T S islets become generally paler, and at the time when blood flow stops, the circulation is momentarily “frozen.” The flow usually resumes and stops intermittently for the next few minutes, and brief, intermittent slowing may occur for the next 20 or 30 minutes. In higher dilutions, there is intermittent slowing of insular circulation, but the flow does not stop. The 1 : 1,000,000 solution has no visible effect. Epinephrine applied locally has effects quite similar to the changes following intravenous injection, but the duration is not as long, usually less than ten minutes in all. L-norepinephrine injected intravenously exerts a greater effect on the arteries and veins of the pancreas than on the arterioles and venules. Within one minute after a dose of 2.4 pg, there is segmental constriction of secondary and tertiary (lobar) arteries, and slight uniform narrowing of the corresponding veins. At three minutes, the circulation in the islets slows temporarily, but the blood flow never stops, and there is no constriction of arterioles, venules or capillaries. By seven minutes, the arterial and venous constriction has disappeared, and the circulation is normal. A dose of 0.8 pg produces similar but less pronounced changes of about the same duration, with slower blood flow in the islets, slight constriction in the secondary lobular arteries, and slow flow in corresponding lobular veins. Injection of 0.4 pg has no visible effects on the pancreatic circulation. Applied locally in solutions of 0.0008% and higher, I-norepinephrine constricts arterioles and venules a s well as arteries and veins, and the circulation in the islets stops momentarily. The duration of action is shorter when the drug is applied locally. Ephedrine injected intravenously causes changes in the insular microcirculation which closely resemble the actions of epinephrine. The respiration is affected also, becoming jerky, deep and rapid. The pulse rate is increased. The circulation in the islets stops completely for less than two minutes. The flow of blood in the islets is slow for the next 15 or 20 minutes, and then it returns to normal. Intermittent slowing and stopping of circulation, as seen with epinephrine, is not observed. Ephedrine applied locally to the islets causes the same effects on pulse and insular microcirculation but has no influence on respiration. Pitressin injected intravenously causes slowing of the flow of blood in the islets and in arterioles and venules, but there is no appreciable vasoconstriction i n these vessels. The pulse rate is slowed. In high concentrations, pallor of the islets is observed. All dilutions tested have some effect on blood flow. In contrast, pitressin applied directly to the pancreas has no apparent effect on the circulation. Insulin, glucagon, hydrocortisone, glucose, and alloxan have no visible effect upon the microcirculation in the islets. The dose of alloxan is sufficient to cause degranulation and vacuolation of the beta cells of the islets, as demonstrated by histologic sections. In the case of glucagon, no histologic changes are evident. Diphenylthiocarbazone injected intravenously has no visible effect on the microcirculation i n the islets, in the concentrations used. About five minutes after injection, the center of the islet begins to turn red. As the color deepens, the periphery turns red also. After about 45 minutes, the color fades gradually, and by 90 minutes, the islets are no longer red. DISCUSSION Our findings with respect to the vascular connections of the islets, and the arrangement of the insular capillaries, agree with those of Brunfeldt, Hunhammar and Skouby (’58). Direct connections of the capillaries of the islets and the capillaries of the acinar parenchyma, as reported by Berg (‘30a, b), Beck and Berg (‘31), Ferner (’52), Wharton (’32) and others, were not observed, either in the injected specimens or in the living pancreas. The flow of blood in the islets of the anesthetized, unstimulated mouse is constant, under the conditions of our observations. This finding does not necessarily mean that blood flow i n the islets of a n unanesthetized, active mouse is constant also. Rather, uninterrupted flow may be only a reflection of the rigid control of the conditions of these observations. The constriction of arterioles and venules, and the transient interruption of in- 122 SRICHITRA C . B U N N A G , SIROTMA B U N N A G A N D N A N C Y E. W A R N E R sular blood flow following the intravenous administration of epinephrine and ephedrine contrasts with the activity of norepinephrine, which has greater effect on arteries and veins, and does not cause the circulation in the islets to stop, when administered intravenously. The hyperglycemic effect of epinephrine and ephedrine is well known (Goodman and Gilman, '55); in contrast, norepinephrine causes only a slight increase in blood sugar. The possibility that the hyperglycemia caused by epinephrine and ephedrine is related to the interruption of insular circulation is suggested by these findings, but proof is lacking at present. The accepted mechanism by which epinephrine causes hyperglycemia is via its glycogenolytic action (Goodman and Gilman, '55). Recently, another component of epinephrine hyperglycemia has been described (Rosenberg and DiStefano, '62), relating to the central nervous system, and it does not seem unreasonable to suppose that still other synergistic mechanisms may exist. The association of diabetes mellitus with pheochromocytoma is of interest. Those tumors which secrete mainly epinephrine may produce diabetes in addition to hypertension, while norepinephrine-secreting tumors are more likely to be associated solely with hypertension (Freedman, Moulton, Rosenheim, Spencer and Willoughby, '58). Some patients are cured of diabetes by removal of the tumor, but in others the diabetes persists. The pathogenesis of the diabetes is not understood, but in the light of the present study, the differential effects of epinephrine and norepinephrine on the microcirculation in the islets of Langerhans could be a factor. Concerning the adjustment of the concentration of insulin in the body under physiological conditions, the evidence available indicates that the concentration of glucose in the blood flowing through the pancreas regulates the rate of insulin secretion (Banting and Gairns, '24; Woerner, '38; Gomori, Friedman and Caldwell, '39; Anderson and Long, '47). When the insular blood flow is interrupted, as by the effect of epinephrine or ephedrine, glucose is prevented from reaching the islets, and the release of insulin could be affected. The action of glucose in release of insulin from the pancreas is not accompanied by a visible vasomotor reaction in the islets. Similarly, the actions of insulin, glucagon and hydrocortisone are not mediated by an acute vasomotor reaction in the islets. Extracts of posterior pituitary are known to cause hyperglycemia (Ingle, '48), and the effect has been ascribed to hepatic glycogenolysis. It is possible that alterations in microcirculation in the islets may have a role in the hyperglycemia. Although alloxan has no acute vasomotor effects as studied by this method, observations at longer intervals are needed to evaluate fully the role of the microvasculature of the islets in genesis and recovery in alloxan diabetes. Diphenylthiocarbazone, the chelating agent which combines with zinc in the islets to form a red complex, is diabetogenic also (Brunfeldt and Skouby, '56), but has no acute vasomotor effects. Increased blood flow in the islets following administration of alloxan and diphenylthiocarbazone has been reported (Brunfeldt, Hunhammar and Skouby, '58). LITERATURE CITED Anderson, E., and J. A. Long 1947 The effect of hyperglycemia on insulin secretion as determined with the isolated rat pancreas in a perfusion apparatus. Endocrinology, 40: 9297. Banting, F. G., and S. Gairns 1924 Factors influencing the production of insulin. Amer. J. Physiol., 68: 24-30. Beck, J. S. 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Friedman and D. W. Caldwell 1939 Beta cell changes i n guinea pig pancreas in relation to blood sugar level. Proc. SOC.Exp. Biol. Med., 41: 567-570. Gomori, G. 1941 Observations with differential stains on human islands of Langerhans. Amer. J. Path., 17: 395-406. Goodman, L. S., and A. Gilman 1955 The Pharmacological Basis of Therapeutics. (2nd ed.) New York, Macmillan Company. Greene, E. C. 1935 Anatomy of the Rat. Trans. Amer. Phil. SOC.,27: 1-370. Gurr, E. 1956 A Practical Manual of Medical and Biological Staining Techniques. New York, Interscience Publishers, Inc. Ingle, D. J. 1948 The production of experimental glycosuria in the rat. In “Recent Progress in Hormone Research. Vol. 11.” (G. Pincus, ed.), Academic Press, Inc., New York. Knisely, M. H. 1954 The fused quartz rod techniaue for transilluminatine. living internal organs in situ for microscopic study. Anat. Rec., 120: 265-275. Knisely, M. H., L. Warner and F. Harding 1960 Ante-mortem settling. Angiology, 11 : 535-588. - I 123 Kiihne, W., and A. Sh. Lea 1882 Beobachtungen iiber die Absonderung des Pankreas. Unters. physiol. Inst. Heidelberg, 2: 4 4 8 4 8 7 . O’Leary, J. L. 1930 An experimental study of the islet cells of the pancreas in vivo. Anat. Rec., 45: 27-58. Opie, E. L. 1903 Disease of the Pancreas. Philadelphia and London, J. B. Lippincott Company. Palmer, A. A. 1959 A study of blood flow in minute vessels of the pancreatic region of the rat with reference to intermittent corpuscular flow i n individual capillaries. Quart. J. Exp. Physiol., 44: 149-159. Rosenberg, F. J., and V. DiStefano 1962 A central nervous system component of epinephrine hyperglycemia. Amer. J. Physiol., 203: 782-788. Wharton, G. K. 1932 The blood supply of the pancreas, with special reference to that of the islands of Langerhans. Anat. Rec., 53: 5581. Woerner, C. A. 1938 Studies of the islands of Langerhans after continuous intravenous injection of dextrose. Ibid., 71: 33-57.