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In vivo fluorescence microscopy of blood flow in mouse pancreatic islets: Adrenergic effects in lean and obese-hyperglycemic mice
MICROVASCULAR
RESEARCH
30, 176-184 (1985)
/n Viva Fluorescence Microscopy of Blood Flow in Mouse Pancreatic Islets: Adrenergic Effects in Lean and ObeseHyperglycemic Mice PAL ROOTH, KJELL GRANKVIST, AND INGE-BERT T~LJEDAL Department of Histology and Cell Biology, University of Vme6, S-901 87 VmeH, Sweden Received December 20, 1983 The microcirculation in the islets of Langerhans was examined by fluorescence microscopy in living mice injected with fluorescent dextran. The islet capillary network was denser and more tortuously arranged in obese-hyperglycemic (oblob) mice than in lean controls. Injection of norepinephrine (0.5-4.0 pg/kg body wt) immediately led to a pronounced inhibition of islet blood flow in oblob mice. In experiments with lean mice less striking effects were seen. With as high a dose as 20 pg norepinephrine/kg body wt only a slight retardation and very brief stop of the flow occurred. The inhibition in oblob mice was blocked by phentolamine, indicating that the norepinephrine-induced inhibition was mediated by a-adrenoceptors. The cy-2-adrenoceptor agonist, clonidine, had no effect on islet blood flow, suggesting that the effect of norepinephrine was due to cY-1-adrenoceptorstimulation. It is concluded that in the living animal norepinephrine inhibits insulin secretion from the pancreas by a twofold mechanism involving inhibition of exocytosis (a-2-receptors on the p-cells) as well as retardation of blood flow (a-l-receptors on blood vessels). o 1985 Academic Press, Inc.
INTRODUCTION When studying the physiological and pharmacological control of insulin secretion, considerable attention has been payed to the responses of the pancreatic p-cells to various agents. Little work has been done on the regulation of blood flow in the endocrine pancreas. It seems conceivable that the rate of hormone delivery to the various target organs is not only controlled by the secretory process as such but also by the rate of blood transport through the islets. In attempts to quantify the pancreatic islet blood flow, some research groups have injected microspheres into living animals and then counted their appearance in islets and exocrine parenchyma (Lifson et al., 1980; Meyer et al., 1982; Jansson and Hellerstrom, 1983). This technique seems valuable, but the static preparations on which the final analysis is made only permit indirect conclusions to be drawn about the dynamic blood flow. Injection of fluorescent substances has been used to visualize the circulation in hamster cheek pouches (Del Maestro et al., 1981a,b), and as reported during the preparation of the present manuscript, in the pancreas (Ohtani, 1983). The purpose of our study was to explore the usefulness of fluorescence microscopy for direct observation of the dynamic circulation in islets in situ. Because previous 176 0026-2862/t% $3.00 Copyright Q 1985 by Academic F’ress, Inc. AU rights of reproduction in any form reserved. F’rinted in U.S.A.
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reports on the effects of adrenergic substances on pancreatic blood flow are somewhat contradictory (Bunnag et al., 1977; Meyer et al., 1982), we used the in situ fluorescence microscopy technique to test the influences of norepinephrine, clonidine, and phentolamine. MATERIALS
AND METHODS
Adult noninbred female oblob mice weighing 45-55 g, and phenotypically normal lean mice weighing 18-25 g were taken from a local colony and starved overnight with free access to water. Several methodological pilot experiments apart, the study comprised more than 40 oblob mice and 20 lean mice; the exact numbers of animals used in pharmacological tests are specified in table and figure legends. The animals were anesthetized by an intraperitoneal injection of pentobarbital (75 mg/kg body wt). Laparotomy was performed through a dorsolateral incision and a lobe of the pancreas was carefully mobilized to the surface of the body. A thin cannula (0.4 mm diameter) was inserted into a tail vein, and, to achieve approximately the same fluorescence in both types of animals, 50 ~1 (oblob mice) or 25 ~1 (lean mice) of 5% fluorescein isothiocyanate (FITC)-dextran (Pharmacia, Uppsala, Sweden) was injected; the molecular weight of FITCdextran was 150,000. The animal under study was then placed on the object stage of a fluorescence microscope equipped with Ploem epi-illumination and a television camera (JVC TH 1700) that made it possible to inspect the islets both via the oculars and on a television monitor (Sony pvm 90-ce). The camera was also connected to a video tape recorder (Sony ~0-2630 U-matic) allowing the experimental results to be carefully analyzed by playback after the experiments were completed. To describe the different flow conditions, terms like “total stop,” “noticeable inhibition,” and “normal circulation” will be used. The total stop was a condition without any detectable movement of corpuscles in the capillaries. Noticeable inhibition refers to a flow condition which differs from total stop and normal circulation in a manner clearly obvious to the observer in that the movement of single blood corpuscles could easily be followed. During normal circulation the movement of corpuscles was blurred because of the much higher flow velocity. After an islet had been identified, various pharmacological agents were injected into a tail vein through the inserted cannula: 50-150 ~1 norepinephrine (0.5-20.0 pg/kg body wt), loo-230 ~1 phentolamine (0.1-1.0 mg/kg body wt), or 70-170 ~1 clonidine (2.0-3.0 pg/kg body wt). The drugs were diluted with physiological saline from pharmaceutical stocks intended for clinical use. Norepinephrine was Noradrenalin (1 mg/ml) from Apoteksbolaget, Sweden; phentolamine was Regitin (10 mg/ml) from Ciba-Geigy; and clonidine was Catapresan (150 ,ug/ml) from Boehringer Ingelheim. The injection time was 5-10 sec. When more than one concentration of a drug was tested in the same animal, injections were spaced by 10 min. RESULTS General The injected fluorescent dextran made the microvessels of the pancreas clearly visible (Fig. 1); islets could be identified with ease at both 130x and 320x
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FE. 1. Fluorescent micrograph of pancreas in a living oblob mouse intravenously injected with FITC-dextran. The light area in the center is an islet contrasting against the darker surrounding of exocrine parenchyma. The tortuously arranged streaks in the islet represent sinusoids containing FITC-dextran. The picture was taken during normal flow conditions. Final magnification about 75 x
magnification. The quality of the television pictures allowed one to see without difficulty how the blood moved in the sinusoids of the islets and in the capillaries of the exocrine parenchyma. In untreated animals there was no visible leakage of FITC-dextran out of the vessels. Differences
between oblob Mouse and Lean Mouse Islets
In both types of mice the islets appeared as round to ovoid light areas against a background of darker exocrine parenchyma. The islet vessels were much wider than those in the acinar tissue. In lean mice the sinusoids had a rather straight arrangement with occasional spirals and bends. The width of the sinusoids was also rather uniform. In comparison with the lean mice, the islets of oblob mice contained a denser network of capillaries, which were more tortuous, wider on an average, and of more variable width. Connecting vessels of capillary size between the endocrine islets and the surrounding exocrine tissue were seen in both types of mice. In some islets of oblob mice direct efferent vessels connecting to larger veins were observed concomitantly with thin efferent capillaries radiating to communicate with the capillary net of the acinar tissue. Leukocyte plugging and spontaneous arrests of flow were not seen to occur in islet vessels of either type of mouse. Islet Blood Flow in oblob Mice after Injection
of Norepinephrine
Injection of 0.5 pg of norepinephrinelkg body wt immediately led to a noticeable inhibition, and even total stop, of flow in the islet sinusoids (Fig. 2). The effects lasted for about 10 set, whereafter the blood flow seemed normal again. With
BLOOD FLOW IN PANCREATIC
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FIG. 2. Fluorescent micrograph as in Fig. 1, except that the circulation is completely inhibited due to infusion of norepinephrine. Note the dark spots representing arrested blood cells in the microvessels of both endocrine and exocrine pancreas. Final magnification about 50 x .
higher dosages of norepinephrine (1 .O, 2.0, or 4.0 pug/kgbody wt), the circulatory arrests became progressively prolonged (Fig. 3). In some of the experiments the blood flow was blocked for up to 35 sec. After the acute standstill the circulation started intermittently for a few seconds, before the flow resumed to normal or, in many cases, developed into a condition observed as hyperemia lasting for 1S-2 min. During the circulatory arrests no signs of platelet aggregation or other evidences of blood clotting were seen. Islet Blood Flow in Lean Mice after Injection of Norepinephrine In some experiments with 0.5 pg norepinephrine/kg body wt a noticeable inhibition or very brief total stop in the circulation was observed in islets of lean mice. In other experiments with the same dose of norepinephrine no effects were seen. At higher dosages (1 .O, 2.0, or 6.0 pug/kgbody wt) norepinephrine had no visible effects on islet circulation in a majority of the experiments (Fig. 4, Table 1). With as much as 20 pg norepinephrine/kg body wt only a slight noticeable inhibition and a very short-lasting total stop of flow was observed. Effects of Phentolamine on Islet Blood Flow in oblob Mice To elucidate the nature of the adrenergic receptors mediating the marked effects of norepinephrine on islet circulation in oblob mice, pretreatment with the LYblocker, phentolamine, was tested. Phentolamine at 1.0 or 0.1 mg/kg body wt completely prevented the circulatory retardation induced by 0.5, 1.0, 2.0, or 4.0 ,ug norepinephrineikg body wt. Phentolamine alone had no effects on islet circulation.
180
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TALJEDAL
26
6
NOREPINEPHRINE
(pg/kg)
3. Effects of norepinephrine injection on the microcirculation in the islets of oblob mice. The time of complete circulatory standstill (open bars) or of any noticeable retardation of flow (stippled bars) is shown as mean values * SEM for 0.5 pg norepinephrine/kg BW (5 experiments/ 5 mice), 1.0 pg norepinephrine/kg BW (4 experiments/4 mice), 2.0 &kg BW (5 experiments/4 mice), and 4 pg norepinephrine/kg BW (3 experiments12 mice). FIG.
Effects of Clonidine on Islet Blood Flow in oblob Mice To test whether the marked effects of norepinephrine on islet circulation in oblob mice were mediated by a-Zreceptors, clonidine was infused and tested for any effect on the microcirculation. This relatively selective cY-Zagonist was administered in dosages between 2 and 30 pg/kg body wt. No effects on islet circulation were noticed. TABLE 1 EFFECTS OF NOREPINEPHRINE ON ISLET BLOOD FLOW IN ob/ob AND LEAN MICE
Norepinephrine infused @g/kg BW) 0.5
1.0 2.0 4.0 6.0
No. of expts with noticeable inhibition/total no. of expts objob
Lean
515 414 515 313
318
116 l/5
o/2
Note. Inhibition is defined as a noticeable retardation of the circulation whether or not a total stop occurred. Altogether, 7 ob/ob mice and 6 lean mice were studied.
BLOOD FLOW IN PANCREATIC
0.5 I .o NOREPINEPHRINE
ISLETS
181
2.0 (pg/kg)
FIG. 4. Effects of norepinephrine injection on the microcirculation in the islets of lean mice. Results are presentedas in Fig. 3 for the minority of lean mice respondingaccording to Table 1.
DISCUSSION The vascular structure of the pancreas has been studied by dye injection techniques (Ktihne and Lea, 1882; Beck and Berg, 1931; Wharton, 1932) and, more recently, by using corrosion casts (Fujita and Murakami, 1973; Bonner-Weir and Orci, 1982; Ohtani, 1983). In most species an insuloacinar portal system exists (Fujita, 1973; Fujita and Murakami, 1973; Daniel and Henderson, 1978; Henderson and Daniel, 1979; Fraser and Henderson, 1980; Ohtani, 1983; Chilvers and Thomas, 1983), supporting the idea of a functional relationship between the endocrine and exocrine pancreas (Kramer and Tan, 1968; Soling and Unger, 1972; Alder and Kern, 1975; Frier et al., 1976; Korc et al., 1981). Our study demonstrates the usefulness of direct fluorescence microscopy to study circulatory events in the endocrine pancreas in vivo. It has been suggested that intermittent islet blood flow may be caused by precapillary smooth muscle sphincters in the acinar tissue and by specialized contractile endothelial cells in islet capillaries (McCuskey and Chapman, 1969). The present experiments gave no indication of such a selective flow regulation between single islet capillaries under control conditions. The blood flow in the individual capillaries of islets and acinar tissue was constant and without intermittent changes in velocity. That islet capillaries have a greater diameter than those in the exocrine pancreas has been reported (Brunfeldt et al., 1958; Henderson and Daniel, 1979; Bonner-Weir and Orci, 1982) and was clearly observed by the present technique. In addition, the islets of oblob mice were easily seen to have a denser network of patent capillaries with larger width as compared with lean mice. A similar observation has been made on static pancreas preparations of animals injected with vasoflavine (Hellerstriim and Hellman, 1961). The increased vascularization might retlect a high rate of oxygen consumption in the islets of oblob mice or an enhanced insulin transport or both. According to measurements in vitro, the endogenous as well as the glucose-stimulated respiration of isolated islets does not seem to be higher in oblob mice than in normal mice (Hellerstrom et al., 1980), suggesting that the increased density of sinusoids is largely related to the increased hormone transport in oblob mice. The circulatory level of insulin in these mice is of the order of 10 times that in normal controls (Westman, 1968). In both types of mice there were direct connecting vessels between islets and exocrine parenchyma indicative of an insuloacinar portal system similar to that observed in previous reports on various species (Fuji& 1973; Fujita and Murakami,
182
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1973; Fraser and Henderson, 1980; Lifson et al., 1980; Chilvers and Thomas, 1983; Ohtani, 1983). Interestingly, in one of our experiments two types of drainage were observed in an oblob mouse islet: in one part of the islet efferent capillary vessels radiated to communicate with the capillary net in the exocrine tissue, while in another part of the same islet there were direct efferent vessels connecting to larger veins. Although these two types of vascular arrangement have been described before, as far as we know they have not been shown to occur simultaneously in one islet. Rappaport et al. (1971) observed a decreased blood flow and insulin output of the whole pancreas in situ during norepinephrine infusion. In contrast, Meyer et al. (1982) reported an increase in total pancreatic blood flow after infusion of norepinephrine. Our study revealed that injection of norepinephrine into oblob mice has marked and prompt inhibitory effects on islet circulation. The effect was dose dependent and, as it was blocked by phentolamine, seemed to be mediated by a-adrenoceptors. The relatively selective a-Zagonist, clonidine, had no visible effect on islet blood flow, suggesting the involvement of a-l-receptors. In contrast, the adrenergic inhibition of insulin release from isolated islets is mediated by a-Zreceptors on the p-cells (Ismail et al., 1983). Perhaps the results of Meyer et al. (1982) are partly influenced by hyperemia similar to the one observed here to follow the acute standstill after norepinephrine injection. In their experiments norepinephrine was infused for 15 min, and then microspheres were injected. The microsphere distribution might then have visualized a condition of a reactive increase in blood flow. The islet circulatory arrest immediately following a bolus injection of norepinephrine is likely to be mediated by the peripheral a-l-receptors. As norepinephrine increases heart rate and cardiac output (Imms et al., 1977; Meyer et al., 1982) and raises arterial blood pressure (Rappaport et al., 1971), the secondary hyperemia may in part be due to cardiac effects of norepinephrine. Bunnag et al. (1977) found that intravenous norepinephrine had a greater constriction effect on the arteries and veins than on the arterioles and venules of the pancreas. In our experiments we have seen inhibition of islet circulation in spite of an apparently normal flow in vessels of arteriolar size, a result suggesting differences in local Aow regulation between the endocrine and exocrine parts of the pancreas. Our technique did not allow a simultaneous study of other parts of the exocrine pancreas than those very close to an islet. The small vessels of the exocrine parenchyma in close vicinity to an islet showed the same responses as the islet vessels to adrenergic stimulation. The oblob mice were found to be more sensitive to norepinephrine than were the lean controls. One explanation could be a difference in the distribution of adrenoceptors. Further experiments are required to elucidate whether analogous differences exist in other organs, which could perhaps be of importance for the pathophysiology of the obese-hyperglycemic syndrome in these mice. ACKNOWLEDGMENTS This work was supported by the Swedish Diabetes Association, The Swedish Medical Research Council (12x-2288), the Magnus Bergwall Foundation, and Novo Industri AB.
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REFERENCES ALDER, G., AND KERN, H. F. (1975). Regulation of exocrine pancreatic secretory process by insulin in vivo. Horm,
Metab.
Res. 7, 290-296.
BECK, J. S. P., AND BERG,B. N. (1931). Circulatory pattern in the islands of Langerhans. Amer. J. Pathol. 7, 31-36. BONNER-WEIR,S., AND ORCI, L. (1982). New perspective on the microvasculature of the islets of Langerhans in the rat. Diabetes 31, 883-889. BRUNFELDT,K., HUNHAMMAR,K., AND SKOUBY,A. P. (1958). Studies on the vascular system of the islets of Langerhans in mice. Acta Endocrinol. (Copenhagen) 29, 475-480. BUNNAG,S. C., WARNER,N. E., AND BUNNAG,S. (1977). Vasomotor reactions in the islets affecting the blood glucose levels. Bibl, Anat. 16, 445-449. CHILVERS,E. R., AND THOMAS,N. W. (1983). The blood supply of the islets of Langerhans in the Mongolian Gerbil. J. Anaf. 136, 339-347. DANIEL, P. M., AND HENDERSON,J. R. (1978). Circulation of the islets of Langerhans. J. Physiol. (London) 275, lOP-1lP. DEL MAESTRO,R. F., BJ~RK, J., AND ARFORS,K.-E. (198la). Increase in microvascular permeability induced by enzymatically generated free radicals. I. In vivo study. Microvasc. Res. 22, 239-254. DEL MAESTRO,R. F., BJ~RK, J., AND ARFORS,K.-E. (198lb). Increase in microvascular permeability induced by enzymatically generated free radicals. II. Role of superoxide anion radicals, hydrogen peroxide, and hydroxyl radical. Microvasc. Res. 22, 255-270. FRASER,P. A., AND HENDERSON, J. R. (1980). The arrangement of endocrine and exocrine pancreatic microcirculation observed in the living rabbit. Q. 1. Exp. Physiol. 65, 151-158. FRIER, B. M., SAUNDERS,J. H. B., WORMSLEY,K. G., AND BOUCHIER,I. A. D. (1976). Exocrine pancreatic function in juvenile-onset diabetes mellitus. Gut 17, 685-691. FUJITA, T. (1973). Insulo-acinar portal system in the horse pancreas. Arch. Histol. Japon. 35, 161171. FUJITA, T., AND MURAKAMI,T. (1973). Microcirculation of monkey pancreas with special reference to insulo-acinar portal systems: A scanning electron microscope study of vascular casts. Arch. Histol.
Japon 35, 255-263.
HELLERSTR~M, C., ANDHELLMAN,B. (l%l). The blood circulation in the islets of Langerhans visualized by the fluorescent dye vasoflavine. Acta Sot. Med. Ups. 66, 88-94. HELLERSTR~M, C., ANDERSSON, A., ANDWELSH,M. (1980). Respiration of the pancreatic p-cell: Effects of glucose and 2-amino-norbomane-2-carboxylic acid. Horm. Metab. Res. suppl. 10, 37-43. HENDERSON, J. R., AND DANIEL, P. M. (1979). A comparative study of portal vessels connecting the endocrine and exocrine pancreas, with a discussion of some functional implications. Q. J. Exp. Physiol.
64, 267-275.
IMMS, F. J., NEAME, R. L. B., AND Powrs, D. A. (1977). Responses of the cardiovascular system of the rat to noradrenaline infusions and their modification by adrenoceptor blocking agents. &it. J. Pharmacol. 60, 115-121. ISMAIL, N. A., EL-DENSHARY,E. S. M., IDAHL, L.-A., LINDSTR~M,P., SEHLIN, J., AND T~LJEDAL, I.-B. (1983). Effects of alpha-adrenoceptor agonists and antagonists on insulin secretion, calcium uptake, and rubidium efflux in mouse pancreatic islets. Acta Physiol. Stand. 118, 167-174. JANSSON,L., AND HELLERSTR~M, C. (1983). Stimulation by glucose of the blood flow to the pancreatic islets of the rat. Diabetologia 25, 45-50. KORC,M., OWERBACH, D., QUINTO,C., ANDRUTTER,W. J. (1981).Pancreatic islet-acinar cell interactions: Amylase messenger RNA levels are determined by insulin. Science (Washington, D.C.) 213, 351353.
KRAMER,M. F., ANDTAN, H. T. (1968). The per&insular acini of the pancreas of the rat. Z. Zel(forsch. Mikroskop. Anat. 86, 163-170. K~HNE, W., AND LEA, A. S. (1882). Beobachtungen iiber die Absonderung des Pankreas. Unters. Physiol.
Inst. Univ. Heidelberg
2, 448-487.
LIFSON, N., KRAMLINGER,K. G., MAYRAND,R. R., AND LENDER,E. J. (1980). Blood flow to the rabbit pancreas with special reference to the islets of Langerhans. Gastroenterology 79, 466-473. MCCUSKEY,R. S., AND CHAPMAN,T. M.(1%9). Microscopy of the living pancreas in situ. Amer. J. Anat.
126, 395-408.
MEYER, H. H., VETTERLEIN,F., SCHMIDT,G., AND HASSELBLATT, A. (1982). Measurement of blood
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AND
TiMEDAL
flow in pancreatic islets of the rat: Effect of isoproterenol and norepinephrine. Amer. J. Physiol. 242, E298-E304. OHTANI, 0. (1983). Microcirculation of the pancreas: A correlative study of intravital microscopy with scanning electron microscopy of vascular casts. Arch. Histol. Japan. 46, 315-325. RAPPAPORT, A. M., KAWAMURA, T., DAVIDSON, J. K., LIN, B. J., OHIRA, S., ZEIGLER, J. A., CODDLING, A. O., HENDERSON, J., AND HAIST, E. (1971). Effects of hormones and of blood flow on isolated pancreas in situ. Amer. J. Physiol. 221, 343-348. SOLING, H. D., AND UNGER, K. 0. (1972). The role of insulin in the regulation of amylase synthesis in the rat pancreas. Eur. J. C/in. Invest. 2, 199-212. WESTMAN, S. (1968). Development of the obese-hyperglycemic syndrome in mice. Diubetologia 4, 141-149. WHARTON, G. R. (1932). The blood supply of the pancreas with special reference to that of the islands of Langerhans. Anat. Rec. 53, 55-81.
RESEARCH
30, 176-184 (1985)
/n Viva Fluorescence Microscopy of Blood Flow in Mouse Pancreatic Islets: Adrenergic Effects in Lean and ObeseHyperglycemic Mice PAL ROOTH, KJELL GRANKVIST, AND INGE-BERT T~LJEDAL Department of Histology and Cell Biology, University of Vme6, S-901 87 VmeH, Sweden Received December 20, 1983 The microcirculation in the islets of Langerhans was examined by fluorescence microscopy in living mice injected with fluorescent dextran. The islet capillary network was denser and more tortuously arranged in obese-hyperglycemic (oblob) mice than in lean controls. Injection of norepinephrine (0.5-4.0 pg/kg body wt) immediately led to a pronounced inhibition of islet blood flow in oblob mice. In experiments with lean mice less striking effects were seen. With as high a dose as 20 pg norepinephrine/kg body wt only a slight retardation and very brief stop of the flow occurred. The inhibition in oblob mice was blocked by phentolamine, indicating that the norepinephrine-induced inhibition was mediated by a-adrenoceptors. The cy-2-adrenoceptor agonist, clonidine, had no effect on islet blood flow, suggesting that the effect of norepinephrine was due to cY-1-adrenoceptorstimulation. It is concluded that in the living animal norepinephrine inhibits insulin secretion from the pancreas by a twofold mechanism involving inhibition of exocytosis (a-2-receptors on the p-cells) as well as retardation of blood flow (a-l-receptors on blood vessels). o 1985 Academic Press, Inc.
INTRODUCTION When studying the physiological and pharmacological control of insulin secretion, considerable attention has been payed to the responses of the pancreatic p-cells to various agents. Little work has been done on the regulation of blood flow in the endocrine pancreas. It seems conceivable that the rate of hormone delivery to the various target organs is not only controlled by the secretory process as such but also by the rate of blood transport through the islets. In attempts to quantify the pancreatic islet blood flow, some research groups have injected microspheres into living animals and then counted their appearance in islets and exocrine parenchyma (Lifson et al., 1980; Meyer et al., 1982; Jansson and Hellerstrom, 1983). This technique seems valuable, but the static preparations on which the final analysis is made only permit indirect conclusions to be drawn about the dynamic blood flow. Injection of fluorescent substances has been used to visualize the circulation in hamster cheek pouches (Del Maestro et al., 1981a,b), and as reported during the preparation of the present manuscript, in the pancreas (Ohtani, 1983). The purpose of our study was to explore the usefulness of fluorescence microscopy for direct observation of the dynamic circulation in islets in situ. Because previous 176 0026-2862/t% $3.00 Copyright Q 1985 by Academic F’ress, Inc. AU rights of reproduction in any form reserved. F’rinted in U.S.A.
BLOOD FLOW IN PANCREATIC
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reports on the effects of adrenergic substances on pancreatic blood flow are somewhat contradictory (Bunnag et al., 1977; Meyer et al., 1982), we used the in situ fluorescence microscopy technique to test the influences of norepinephrine, clonidine, and phentolamine. MATERIALS
AND METHODS
Adult noninbred female oblob mice weighing 45-55 g, and phenotypically normal lean mice weighing 18-25 g were taken from a local colony and starved overnight with free access to water. Several methodological pilot experiments apart, the study comprised more than 40 oblob mice and 20 lean mice; the exact numbers of animals used in pharmacological tests are specified in table and figure legends. The animals were anesthetized by an intraperitoneal injection of pentobarbital (75 mg/kg body wt). Laparotomy was performed through a dorsolateral incision and a lobe of the pancreas was carefully mobilized to the surface of the body. A thin cannula (0.4 mm diameter) was inserted into a tail vein, and, to achieve approximately the same fluorescence in both types of animals, 50 ~1 (oblob mice) or 25 ~1 (lean mice) of 5% fluorescein isothiocyanate (FITC)-dextran (Pharmacia, Uppsala, Sweden) was injected; the molecular weight of FITCdextran was 150,000. The animal under study was then placed on the object stage of a fluorescence microscope equipped with Ploem epi-illumination and a television camera (JVC TH 1700) that made it possible to inspect the islets both via the oculars and on a television monitor (Sony pvm 90-ce). The camera was also connected to a video tape recorder (Sony ~0-2630 U-matic) allowing the experimental results to be carefully analyzed by playback after the experiments were completed. To describe the different flow conditions, terms like “total stop,” “noticeable inhibition,” and “normal circulation” will be used. The total stop was a condition without any detectable movement of corpuscles in the capillaries. Noticeable inhibition refers to a flow condition which differs from total stop and normal circulation in a manner clearly obvious to the observer in that the movement of single blood corpuscles could easily be followed. During normal circulation the movement of corpuscles was blurred because of the much higher flow velocity. After an islet had been identified, various pharmacological agents were injected into a tail vein through the inserted cannula: 50-150 ~1 norepinephrine (0.5-20.0 pg/kg body wt), loo-230 ~1 phentolamine (0.1-1.0 mg/kg body wt), or 70-170 ~1 clonidine (2.0-3.0 pg/kg body wt). The drugs were diluted with physiological saline from pharmaceutical stocks intended for clinical use. Norepinephrine was Noradrenalin (1 mg/ml) from Apoteksbolaget, Sweden; phentolamine was Regitin (10 mg/ml) from Ciba-Geigy; and clonidine was Catapresan (150 ,ug/ml) from Boehringer Ingelheim. The injection time was 5-10 sec. When more than one concentration of a drug was tested in the same animal, injections were spaced by 10 min. RESULTS General The injected fluorescent dextran made the microvessels of the pancreas clearly visible (Fig. 1); islets could be identified with ease at both 130x and 320x
178
ROOTH,
GRANKVIST,
AND
TiLJEDAL
FE. 1. Fluorescent micrograph of pancreas in a living oblob mouse intravenously injected with FITC-dextran. The light area in the center is an islet contrasting against the darker surrounding of exocrine parenchyma. The tortuously arranged streaks in the islet represent sinusoids containing FITC-dextran. The picture was taken during normal flow conditions. Final magnification about 75 x
magnification. The quality of the television pictures allowed one to see without difficulty how the blood moved in the sinusoids of the islets and in the capillaries of the exocrine parenchyma. In untreated animals there was no visible leakage of FITC-dextran out of the vessels. Differences
between oblob Mouse and Lean Mouse Islets
In both types of mice the islets appeared as round to ovoid light areas against a background of darker exocrine parenchyma. The islet vessels were much wider than those in the acinar tissue. In lean mice the sinusoids had a rather straight arrangement with occasional spirals and bends. The width of the sinusoids was also rather uniform. In comparison with the lean mice, the islets of oblob mice contained a denser network of capillaries, which were more tortuous, wider on an average, and of more variable width. Connecting vessels of capillary size between the endocrine islets and the surrounding exocrine tissue were seen in both types of mice. In some islets of oblob mice direct efferent vessels connecting to larger veins were observed concomitantly with thin efferent capillaries radiating to communicate with the capillary net of the acinar tissue. Leukocyte plugging and spontaneous arrests of flow were not seen to occur in islet vessels of either type of mouse. Islet Blood Flow in oblob Mice after Injection
of Norepinephrine
Injection of 0.5 pg of norepinephrinelkg body wt immediately led to a noticeable inhibition, and even total stop, of flow in the islet sinusoids (Fig. 2). The effects lasted for about 10 set, whereafter the blood flow seemed normal again. With
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FIG. 2. Fluorescent micrograph as in Fig. 1, except that the circulation is completely inhibited due to infusion of norepinephrine. Note the dark spots representing arrested blood cells in the microvessels of both endocrine and exocrine pancreas. Final magnification about 50 x .
higher dosages of norepinephrine (1 .O, 2.0, or 4.0 pug/kgbody wt), the circulatory arrests became progressively prolonged (Fig. 3). In some of the experiments the blood flow was blocked for up to 35 sec. After the acute standstill the circulation started intermittently for a few seconds, before the flow resumed to normal or, in many cases, developed into a condition observed as hyperemia lasting for 1S-2 min. During the circulatory arrests no signs of platelet aggregation or other evidences of blood clotting were seen. Islet Blood Flow in Lean Mice after Injection of Norepinephrine In some experiments with 0.5 pg norepinephrine/kg body wt a noticeable inhibition or very brief total stop in the circulation was observed in islets of lean mice. In other experiments with the same dose of norepinephrine no effects were seen. At higher dosages (1 .O, 2.0, or 6.0 pug/kgbody wt) norepinephrine had no visible effects on islet circulation in a majority of the experiments (Fig. 4, Table 1). With as much as 20 pg norepinephrine/kg body wt only a slight noticeable inhibition and a very short-lasting total stop of flow was observed. Effects of Phentolamine on Islet Blood Flow in oblob Mice To elucidate the nature of the adrenergic receptors mediating the marked effects of norepinephrine on islet circulation in oblob mice, pretreatment with the LYblocker, phentolamine, was tested. Phentolamine at 1.0 or 0.1 mg/kg body wt completely prevented the circulatory retardation induced by 0.5, 1.0, 2.0, or 4.0 ,ug norepinephrineikg body wt. Phentolamine alone had no effects on islet circulation.
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26
6
NOREPINEPHRINE
(pg/kg)
3. Effects of norepinephrine injection on the microcirculation in the islets of oblob mice. The time of complete circulatory standstill (open bars) or of any noticeable retardation of flow (stippled bars) is shown as mean values * SEM for 0.5 pg norepinephrine/kg BW (5 experiments/ 5 mice), 1.0 pg norepinephrine/kg BW (4 experiments/4 mice), 2.0 &kg BW (5 experiments/4 mice), and 4 pg norepinephrine/kg BW (3 experiments12 mice). FIG.
Effects of Clonidine on Islet Blood Flow in oblob Mice To test whether the marked effects of norepinephrine on islet circulation in oblob mice were mediated by a-Zreceptors, clonidine was infused and tested for any effect on the microcirculation. This relatively selective cY-Zagonist was administered in dosages between 2 and 30 pg/kg body wt. No effects on islet circulation were noticed. TABLE 1 EFFECTS OF NOREPINEPHRINE ON ISLET BLOOD FLOW IN ob/ob AND LEAN MICE
Norepinephrine infused @g/kg BW) 0.5
1.0 2.0 4.0 6.0
No. of expts with noticeable inhibition/total no. of expts objob
Lean
515 414 515 313
318
116 l/5
o/2
Note. Inhibition is defined as a noticeable retardation of the circulation whether or not a total stop occurred. Altogether, 7 ob/ob mice and 6 lean mice were studied.
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2.0 (pg/kg)
FIG. 4. Effects of norepinephrine injection on the microcirculation in the islets of lean mice. Results are presentedas in Fig. 3 for the minority of lean mice respondingaccording to Table 1.
DISCUSSION The vascular structure of the pancreas has been studied by dye injection techniques (Ktihne and Lea, 1882; Beck and Berg, 1931; Wharton, 1932) and, more recently, by using corrosion casts (Fujita and Murakami, 1973; Bonner-Weir and Orci, 1982; Ohtani, 1983). In most species an insuloacinar portal system exists (Fujita, 1973; Fujita and Murakami, 1973; Daniel and Henderson, 1978; Henderson and Daniel, 1979; Fraser and Henderson, 1980; Ohtani, 1983; Chilvers and Thomas, 1983), supporting the idea of a functional relationship between the endocrine and exocrine pancreas (Kramer and Tan, 1968; Soling and Unger, 1972; Alder and Kern, 1975; Frier et al., 1976; Korc et al., 1981). Our study demonstrates the usefulness of direct fluorescence microscopy to study circulatory events in the endocrine pancreas in vivo. It has been suggested that intermittent islet blood flow may be caused by precapillary smooth muscle sphincters in the acinar tissue and by specialized contractile endothelial cells in islet capillaries (McCuskey and Chapman, 1969). The present experiments gave no indication of such a selective flow regulation between single islet capillaries under control conditions. The blood flow in the individual capillaries of islets and acinar tissue was constant and without intermittent changes in velocity. That islet capillaries have a greater diameter than those in the exocrine pancreas has been reported (Brunfeldt et al., 1958; Henderson and Daniel, 1979; Bonner-Weir and Orci, 1982) and was clearly observed by the present technique. In addition, the islets of oblob mice were easily seen to have a denser network of patent capillaries with larger width as compared with lean mice. A similar observation has been made on static pancreas preparations of animals injected with vasoflavine (Hellerstriim and Hellman, 1961). The increased vascularization might retlect a high rate of oxygen consumption in the islets of oblob mice or an enhanced insulin transport or both. According to measurements in vitro, the endogenous as well as the glucose-stimulated respiration of isolated islets does not seem to be higher in oblob mice than in normal mice (Hellerstrom et al., 1980), suggesting that the increased density of sinusoids is largely related to the increased hormone transport in oblob mice. The circulatory level of insulin in these mice is of the order of 10 times that in normal controls (Westman, 1968). In both types of mice there were direct connecting vessels between islets and exocrine parenchyma indicative of an insuloacinar portal system similar to that observed in previous reports on various species (Fuji& 1973; Fujita and Murakami,
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1973; Fraser and Henderson, 1980; Lifson et al., 1980; Chilvers and Thomas, 1983; Ohtani, 1983). Interestingly, in one of our experiments two types of drainage were observed in an oblob mouse islet: in one part of the islet efferent capillary vessels radiated to communicate with the capillary net in the exocrine tissue, while in another part of the same islet there were direct efferent vessels connecting to larger veins. Although these two types of vascular arrangement have been described before, as far as we know they have not been shown to occur simultaneously in one islet. Rappaport et al. (1971) observed a decreased blood flow and insulin output of the whole pancreas in situ during norepinephrine infusion. In contrast, Meyer et al. (1982) reported an increase in total pancreatic blood flow after infusion of norepinephrine. Our study revealed that injection of norepinephrine into oblob mice has marked and prompt inhibitory effects on islet circulation. The effect was dose dependent and, as it was blocked by phentolamine, seemed to be mediated by a-adrenoceptors. The relatively selective a-Zagonist, clonidine, had no visible effect on islet blood flow, suggesting the involvement of a-l-receptors. In contrast, the adrenergic inhibition of insulin release from isolated islets is mediated by a-Zreceptors on the p-cells (Ismail et al., 1983). Perhaps the results of Meyer et al. (1982) are partly influenced by hyperemia similar to the one observed here to follow the acute standstill after norepinephrine injection. In their experiments norepinephrine was infused for 15 min, and then microspheres were injected. The microsphere distribution might then have visualized a condition of a reactive increase in blood flow. The islet circulatory arrest immediately following a bolus injection of norepinephrine is likely to be mediated by the peripheral a-l-receptors. As norepinephrine increases heart rate and cardiac output (Imms et al., 1977; Meyer et al., 1982) and raises arterial blood pressure (Rappaport et al., 1971), the secondary hyperemia may in part be due to cardiac effects of norepinephrine. Bunnag et al. (1977) found that intravenous norepinephrine had a greater constriction effect on the arteries and veins than on the arterioles and venules of the pancreas. In our experiments we have seen inhibition of islet circulation in spite of an apparently normal flow in vessels of arteriolar size, a result suggesting differences in local Aow regulation between the endocrine and exocrine parts of the pancreas. Our technique did not allow a simultaneous study of other parts of the exocrine pancreas than those very close to an islet. The small vessels of the exocrine parenchyma in close vicinity to an islet showed the same responses as the islet vessels to adrenergic stimulation. The oblob mice were found to be more sensitive to norepinephrine than were the lean controls. One explanation could be a difference in the distribution of adrenoceptors. Further experiments are required to elucidate whether analogous differences exist in other organs, which could perhaps be of importance for the pathophysiology of the obese-hyperglycemic syndrome in these mice. ACKNOWLEDGMENTS This work was supported by the Swedish Diabetes Association, The Swedish Medical Research Council (12x-2288), the Magnus Bergwall Foundation, and Novo Industri AB.
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REFERENCES ALDER, G., AND KERN, H. F. (1975). Regulation of exocrine pancreatic secretory process by insulin in vivo. Horm,
Metab.
Res. 7, 290-296.
BECK, J. S. P., AND BERG,B. N. (1931). Circulatory pattern in the islands of Langerhans. Amer. J. Pathol. 7, 31-36. BONNER-WEIR,S., AND ORCI, L. (1982). New perspective on the microvasculature of the islets of Langerhans in the rat. Diabetes 31, 883-889. BRUNFELDT,K., HUNHAMMAR,K., AND SKOUBY,A. P. (1958). Studies on the vascular system of the islets of Langerhans in mice. Acta Endocrinol. (Copenhagen) 29, 475-480. BUNNAG,S. C., WARNER,N. E., AND BUNNAG,S. (1977). Vasomotor reactions in the islets affecting the blood glucose levels. Bibl, Anat. 16, 445-449. CHILVERS,E. R., AND THOMAS,N. W. (1983). The blood supply of the islets of Langerhans in the Mongolian Gerbil. J. Anaf. 136, 339-347. DANIEL, P. M., AND HENDERSON,J. R. (1978). Circulation of the islets of Langerhans. J. Physiol. (London) 275, lOP-1lP. DEL MAESTRO,R. F., BJ~RK, J., AND ARFORS,K.-E. (198la). Increase in microvascular permeability induced by enzymatically generated free radicals. I. In vivo study. Microvasc. Res. 22, 239-254. DEL MAESTRO,R. F., BJ~RK, J., AND ARFORS,K.-E. (198lb). Increase in microvascular permeability induced by enzymatically generated free radicals. II. Role of superoxide anion radicals, hydrogen peroxide, and hydroxyl radical. Microvasc. Res. 22, 255-270. FRASER,P. A., AND HENDERSON, J. R. (1980). The arrangement of endocrine and exocrine pancreatic microcirculation observed in the living rabbit. Q. 1. Exp. Physiol. 65, 151-158. FRIER, B. M., SAUNDERS,J. H. B., WORMSLEY,K. G., AND BOUCHIER,I. A. D. (1976). Exocrine pancreatic function in juvenile-onset diabetes mellitus. Gut 17, 685-691. FUJITA, T. (1973). Insulo-acinar portal system in the horse pancreas. Arch. Histol. Japon. 35, 161171. FUJITA, T., AND MURAKAMI,T. (1973). Microcirculation of monkey pancreas with special reference to insulo-acinar portal systems: A scanning electron microscope study of vascular casts. Arch. Histol.
Japon 35, 255-263.
HELLERSTR~M, C., ANDHELLMAN,B. (l%l). The blood circulation in the islets of Langerhans visualized by the fluorescent dye vasoflavine. Acta Sot. Med. Ups. 66, 88-94. HELLERSTR~M, C., ANDERSSON, A., ANDWELSH,M. (1980). Respiration of the pancreatic p-cell: Effects of glucose and 2-amino-norbomane-2-carboxylic acid. Horm. Metab. Res. suppl. 10, 37-43. HENDERSON, J. R., AND DANIEL, P. M. (1979). A comparative study of portal vessels connecting the endocrine and exocrine pancreas, with a discussion of some functional implications. Q. J. Exp. Physiol.
64, 267-275.
IMMS, F. J., NEAME, R. L. B., AND Powrs, D. A. (1977). Responses of the cardiovascular system of the rat to noradrenaline infusions and their modification by adrenoceptor blocking agents. &it. J. Pharmacol. 60, 115-121. ISMAIL, N. A., EL-DENSHARY,E. S. M., IDAHL, L.-A., LINDSTR~M,P., SEHLIN, J., AND T~LJEDAL, I.-B. (1983). Effects of alpha-adrenoceptor agonists and antagonists on insulin secretion, calcium uptake, and rubidium efflux in mouse pancreatic islets. Acta Physiol. Stand. 118, 167-174. JANSSON,L., AND HELLERSTR~M, C. (1983). Stimulation by glucose of the blood flow to the pancreatic islets of the rat. Diabetologia 25, 45-50. KORC,M., OWERBACH, D., QUINTO,C., ANDRUTTER,W. J. (1981).Pancreatic islet-acinar cell interactions: Amylase messenger RNA levels are determined by insulin. Science (Washington, D.C.) 213, 351353.
KRAMER,M. F., ANDTAN, H. T. (1968). The per&insular acini of the pancreas of the rat. Z. Zel(forsch. Mikroskop. Anat. 86, 163-170. K~HNE, W., AND LEA, A. S. (1882). Beobachtungen iiber die Absonderung des Pankreas. Unters. Physiol.
Inst. Univ. Heidelberg
2, 448-487.
LIFSON, N., KRAMLINGER,K. G., MAYRAND,R. R., AND LENDER,E. J. (1980). Blood flow to the rabbit pancreas with special reference to the islets of Langerhans. Gastroenterology 79, 466-473. MCCUSKEY,R. S., AND CHAPMAN,T. M.(1%9). Microscopy of the living pancreas in situ. Amer. J. Anat.
126, 395-408.
MEYER, H. H., VETTERLEIN,F., SCHMIDT,G., AND HASSELBLATT, A. (1982). Measurement of blood
184
ROOTH,
GRANKVIST,
AND
TiMEDAL
flow in pancreatic islets of the rat: Effect of isoproterenol and norepinephrine. Amer. J. Physiol. 242, E298-E304. OHTANI, 0. (1983). Microcirculation of the pancreas: A correlative study of intravital microscopy with scanning electron microscopy of vascular casts. Arch. Histol. Japan. 46, 315-325. RAPPAPORT, A. M., KAWAMURA, T., DAVIDSON, J. K., LIN, B. J., OHIRA, S., ZEIGLER, J. A., CODDLING, A. O., HENDERSON, J., AND HAIST, E. (1971). Effects of hormones and of blood flow on isolated pancreas in situ. Amer. J. Physiol. 221, 343-348. SOLING, H. D., AND UNGER, K. 0. (1972). The role of insulin in the regulation of amylase synthesis in the rat pancreas. Eur. J. C/in. Invest. 2, 199-212. WESTMAN, S. (1968). Development of the obese-hyperglycemic syndrome in mice. Diubetologia 4, 141-149. WHARTON, G. R. (1932). The blood supply of the pancreas with special reference to that of the islands of Langerhans. Anat. Rec. 53, 55-81.