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MICROSCOPY RESEARCH AND TECHNIQUE 37:497–508 (1997)
Blood Flow Patterns in the Rat Pancreas: A Simulative
Demonstration by Injection Replication and Scanning
Electron Microscopy
TAKURO MURAKAMI,1* TSUYOSHI MIYAKE,2 MARI TSUBOUCHI,1 YUTAKA TSUBOUCHI,1 AIJI OHTSUKA,1
AND TSUNEO FUJITA3
1Department
2Department
3Department
of Anatomy, Okayama University School of Medicine, Okayama, 700 Japan
of Internal Medicine, Okayama University School of Medicine, Okayama, 700 Japan
of Anatomy, Niigata University School of Medicine, Niigata, 951 Japan
KEY WORDS
pancreas; microvascular architecture; microcirculation; corrosion casting; scanning electron microscopy
ABSTRACT
Scanning electron microscopy of vascular casts prepared by arterial injections of
intentionally reduced amounts of resin showed that in the rat pancreas, the casting medium fills
blood capillaries in the endocrine islets more promptly than those in the exocrine lobules and
secretory ducts. Furthermore, the exocrine lobules containing endocrine islets allowed a more rapid
resin flow through the insulo-acinar portal route than those lobules lacking an islet. The capillaries
of secretory ducts were the last portions to be filled with resin. Since the resin used in this study was
as viscous as blood and injected under a physiological pressure, the microcirculatory patterns
demonstrated by the present method reflect the physiological flow pattern of blood in the pancreas.
Microsc. Res. Tech. 37:497–508, 1997 r 1997 Wiley-Liss, Inc.
INTRODUCTION
Complete or sufficient injection of low viscosity casting media into arteries reproduces the whole extent of
blood vascular beds, including thick venous portions
(Murakami, 1971; Murakami et al., 1973). Incomplete
or insufficient injection of such media into arteries
partially reproduces the vascular beds; the arteriolecapillary, capillary-venular or venous systems remain
unfilled (Murakami et al., 1983). Similar injection into
veins can reproduce the venous or veno-capillary system (Murakami et al., 1983). Thorough injection into
veins is not suited for casting a whole blood vascular
bed, since it causes rupture on the capillary level
(Murakami et al., 1983). A combination of adequate
casting methods, both complete and partial, thus, allowed a precise scanning electron microscopic analysis
of the fine vascular arrangement or microcirculatory
patterns in the kidney, cerebral hypophysis and other
organs (Murakami, 1971, 1972; Murakami et al., 1983,
1987, 1993a), including the pancreas (Fujita and Murakami, 1973; Murakami and Fujita, 1992; Murakami
et al., 1992, 1993b, 1994).
This paper describes some findings in the pancreas,
especially those obtained by complete and incomplete
arterial injections (Miyake et al., 1992). The target
organ of the present study is the rat pancreas, since its
microcirculatory pattern has raised much controversy
and discussion (Bonner-Weir and Orci, 1982; Ohtani et
al., 1986), including a complicated insulo-acinar portal
r 1997 WILEY-LISS, INC.
system which additionally has separate or collateral
venous drainage (Miyake et al., 1992; Murakami and
Fujita, 1992).
MATERIALS AND METHODS
Fourteen adult male Wistar rats weighing 350–360 g
were anesthetized with ethyl ether, and their thoracic
aorta was cannulated at the level of the seventh
thoracic vertebra. The animal’s circulatory system
was then rinsed by perfusion with Ringer solution
(15.0–20.0 ml) and injected with a semi-polymerized
and diluted low viscosity methacrylate casting medium
(Murakami, 1971; Murakami et al., 1973) at an injection pressure of 120–130 mm Hg. As a substitute for
this laboratory-prepared medium, a commercially available casting medium (Mercox 2R or 2B, Oken shoji,
Tokyo, Japan) was sometimes used. This medium
was also diluted with 20–30% monomeric methyl
methacrylate (Katayama Kagaku, Osaka, Japan)
prior to the injection. The casting media used in
the present study were thus as viscous as blood (or
had a viscosity of about 4.0 centipoise at room temperature).
*Correspondence to: Prof. Takuro Murakami, Section of Human Morphology,
Department of Anatomy, Okayama University School of Medicine, 2–5–1 Shikatacho, Okayama 700 Japan.
Received 20 September 1994; revised 6 April 1995; accepted 31 May 1995.
498
T. MURAKAMI ET AL.
Fig. 1. Low power scanning electron micrograph of the thoroughly
replicated blood vascular bed of the adult rat pancreas (20 ml resin
injection through the thoracic aorta). Complete injection of low
viscosity casting medium through the thoracic aorta reproduces the
whole extent of the pancreatic blood vascular bed. BD, common bile
duct; EL, exocrine lobule. Bar, 1.0 mm.
MICROCIRCULATORY PATTERNS IN THE RAT PANCREAS
Fig. 2. Thoroughly replicated blood vascular bed of the adult rat
pancreas. The pancreatic blood vascular bed mainly consists of the
capillary networks of the exocrine lobules (EL), endocrine islets (EI
and II) and secretory ducts (SD). The endocrine islets are either
499
embedded as the intralobular islets (II) in the exocrine lobules, or as
the interlobular (extralobular) islets (EI) in the interlobular tissue
spaces or along the secretory ducts. A part of this figure was shown
elsewhere (Miyake et al., 1992). Bar, 500 µm.
500
T. MURAKAMI ET AL.
Fig. 3. Two exocrine lobules (EL1 and EL2) isolated from a
thoroughly replicated specimen. The EL1 lobule contains an intralobular islet (II). This islet issues no insulo-venous efferent vessel, but
emits some marked insulo-acinar portal vessels (arrowheads) directly
draining into the lobular capillaries. The EL1 lobule has thus a dual
blood supply: by the portal vessels and by the acinar branches (la) of
the lobular artery. The EL2 lobule contains no islet. IA, interlobular
artery; IV, interlobular vein; SD, secretory duct; ia, islet afferent
artery (insular branch of the lobular artery). Bar, 100 µm.
The amount of the casting medium used for injection
was changed as follows: 17.0–20.0 ml of the casting
medium was perfused in four rats; 8.0–11.0 ml in four
rats; 3.0–6.0 ml in four rats; 0.5–1.0 ml in the remaining two rats. In some animals of the latter three groups,
the hepatic portal vein was cut off closely below the
portal spiral valve (Booz, 1964) prior to the perfusion
with Ringer solution.
The resin-injected animals were placed for 1 hour in a
hot water bath (60°C), and then the pancreas was
isolated ‘‘en bloc’’ together with the liver, stomach,
duodenum, spleen and kidneys. The isolated organs
were immersed in a hot 10% NaOH solution (60°C)
overnight, and washed for 8 hours in running tap water.
This NaOH treatment followed by washing in tap water
was repeated several times.
The vascular casts of the pancreas and other abdominal organs were frozen in distilled water, freezedried, and dissected with forceps or needles. The pan-
creatic casts were then microdissected with sharpened forceps and needles under a binocular light
microscope, stained with osmium vapor (Murakami
et al., 1973), coated with gold in a vacuum evaporator,
and observed with a scanning electron microscope
(S-2300, Hitachi) using an acceleration voltage of
5 KV.
RESULTS
The injection of 17.0–20.0 ml resin produced
complete vascular casts of the kidney, stomach, duodenum, spleen, pancreas and liver. Incomplete resin
injection allowed only partial replication of the blood
vascular beds of these organs. In general, injection
of 0.5–1.0 ml resin reproduced only the arterial systems in the duodenum and spleen as well as arterial
and arterio-capillary systems of the gastric mucosa.
MICROCIRCULATORY PATTERNS IN THE RAT PANCREAS
501
Fig. 4. A lobule (EL) containing an intralobular islet (II). This
lobule is supplied both by the insulo-acinar portal vessels (arrowheads) and by the acinar branch (la) of the lobular artery. In addition
to the portal vessels, the islet issues a collateral venous (insulo-
venous) efferent vessel (iv) draining into the lobular vein. IA, interlobular artery; IV, interlobular vein; SD, secretory duct; ia, islet afferent
artery. A part of this figure was shown elsewhere (Miyake et al., 1992).
Bar, 150 µm.
In the kidney, the glomeruli were reproduced almost completely by the 1.0 ml resin injection. The
3.0–6.0 ml resin injection sufficiently reproduced
the arterial systems of the duodenum and spleen,
and the capillary plexus of the gastric mucosa. The
kidney glomeruli, including their efferent vessels,
were completely reproduced by the 3.0–6.0 ml resin
injection. When 8.0–11.0 ml of resin was used for
injection, the vascular plexuses of the stomach and
kidneys were thoroughly reproduced, though those of
the duodenum and spleen were insufficiently replicated.
502
T. MURAKAMI ET AL.
Fig. 5. An isolated interlobular islet (EI). The interlobular islet has
a fine (capsular) capillary meshwork (arrowheads), which is homologous with the lobular capillary network. IA, interlobular artery; IV,
interlobular vein; SD, secretory duct; ia, islet afferent artery; iv, islet
efferent vein (insulo-venous efferent vein). Bar, 100 µm.
In the thoroughly replicated specimens (i.e. when
injected with 17–20 ml of resin), it was clearly confirmed that the rat pancreas contains a rich blood
vascular bed which mainly consists of a lobular capillary network supplying the exocrine lobules, insular
capillary networks supplying the endocrine islets of
Langerhans, and ductal capillary networks supplying
the pancreatic ducts (Figs. 1, 2).
The capillary networks of the endocrine islets were
more or less conglomerated. The islets thus identifiable
in the cast specimens were observed as intralobular
islets embedded in the lobular capillary network (Figs.
2–4), or as interlobular (extralobular) islets located
among the lobular capillary networks or along a ductal
capillary plexus (Figs. 2, 5).
The intralobular islets issued insulo-acinar portal
vessels draining into the lobular capillary networks
(Figs. 3, 4). These intralobular islets were distributed in
relatively small or thin lobules, so that they usually
issued some collateral venous (insulo-venous) efferent
Fig. 6. Incompletely replicated adult rat pancreas (1.0 ml resin
injection through the thoracic aorta). The capillary networks of
the islets (arrowheads) are reproduced, whereas those of the lobules
and secretory ducts are not replicated. IA, interlobular artery. Bar,
300 µm.
MICROCIRCULATORY PATTERNS IN THE RAT PANCREAS
503
Fig. 7. An intralobular islet (II) isolated from the cast prepared by
1.0 ml resin injection. In the islet, resin filling begins from the
superficial aspects. The islet efferent vein (iv) arises from the deep
(thick arrow) and superficial (thin arrow) efferent rootlets in the islet.
Arrowhead indicates an insulo-acinar portal vessel. IA, interlobular
artery; ia, islet afferent artery. Bar, 100 µm.
vessels directly draining into the lobular veins (Fig. 4).
The interlobular islets issued only venous (insulovenous) efferent vessels which drained into the ductal
or interlobular veins (Fig. 5). In this manner, many
exocrine lobules contained one or more well-distinguishable islets, and received dual blood supply from the
islets via the insulo-acinar portal vessels and from the
acinar arteries.
With the 0.5–1.0 ml resin injection, the insular
capillary networks were almost sufficiently reproduced,
whereas the capillary networks of exocrine lobules and
ducts remained unfilled (Fig. 6). The insulo-venous
efferent and insulo-acinar portal vessels of the islets
were insufficiently replicated with the 0.5–1.0 ml resin
injection. With this injection, however, it was clearly
noted that the arterially injected resin reached the
islets from their superficial aspects, and that the resin
after reaching the islets moved into the insulo-acinar or
insulo-venous efferent vessels from their superficial
and deep aspects (Fig. 7).
With the 3.0–6.0 ml resin injection, the insular
capillary networks were completely reproduced, whereas
Fig. 8. Incompletely replicated rat pancreas (5.0 ml resin injection). The islets (arrowheads) and their venous efferent (insulovenous) systems are promptly filled with resin, so that the related
systemic venous vessels (V), including the interlobular veins (IV), are
also replicated. IA, interlobular artery. Bar, 500 µm.
the capillary networks of exocrine lobules and ducts
still remained unfilled (Figs. 8–10). The insulo-venous
efferent and insulo-acinar portal vessels of the islets
were sufficiently replicated with this injection. The
venous branches continuous with the insulo-venous
efferent vessels as well as the lobular capillaries supplied by the insulo-acinar portal vessels were also
partially replicated (Fig. 9).
504
T. MURAKAMI ET AL.
Fig. 9. An intralobular islet (II) and its efferent vessels replicated by 5.0 ml resin injection (closer view of a part of Figure 8).
The insulo-venous efferent vessels (iv) are sufficiently reproduced together with their connecting lobular or interlobular veins (V). Replication
of the insulo-acinar portal system is insufficient; the lobular capillaries continuous with the portal vessel (arrowhead) are not reproduced. Bar,
100 µm.
With the 8.0–11.0 ml resin injection, the islets
and their insulo-venous efferent and insulo-acinar
portal vessels were sufficiently reproduced together
with the lobular capillaries directly supplied by
the insulo-acinar portal vessels (Figs. 11, 12). With
this injection, replication of the lobular capillaries
supplied by the acinar arteries was still insufficient;
the same was true for the ductal capillary plexus.
The venous efferent vessels of the islets were usually
filled with larger amount of resin than insulo-acinar
portal vessels, being sufficiently reproduced together
with their connecting intralobular or interlobular
veins. In these veins, some other peripheral branches
were usually reproduced by the retrograde flow of
resin (Fig. 11). This back flow of resin into the peripheral veins typically occurred in specimens in
which the hepatic portal vein was neither cut nor
opened.
Replication of the ductal capillary networks was
insufficient with the 8.0–11.0 ml resin injection. Suffi-
cient casting of these ductal networks was noted in the
17.0–20.0 ml resin injections (Figs. 3–5).
DISCUSSION
The present results confirm that thoroughly replicated specimens are useful for a precise morphological
analysis of blood vascular bed under the scanning
electron microscope. Moreover, they indicate that incompletely replicated specimens (specimens prepared by
injections with intentionally reduced amounts of resin)
are useful to study the inflow modes of the casting
medium into the blood vascular bed as well as its
outflow modes from the vasculature.
Our casting medium was as viscous as blood
and perfused under a physiological pressure. Our previous experiments by intravital microscopy using the
mesenterium of ethyl ether-anesthetized adult rats
have shown that our medium flows through mesenterial capillaries in a fashion similar to that of blood
MICROCIRCULATORY PATTERNS IN THE RAT PANCREAS
Fig. 10. An interlobular or periductal islet (EI) replicated by 6.0 ml
resin injection. The islet is sufficiently reproduced, whereas the
secretory duct (SD) is replicated partially. IA, interlobular artery; IV,
interlobular vein; ia, islet afferent artery; iv, islet efferent vein. Bar,
200 µm.
(Miyake et al., 1992). These data indicate that our
vascular casts prepared by injections of intentionally
reduced amounts of resin through arteries properly
reflect the actual blood flow patterns, and provide
important, additional information concerning the dynamic flow of blood. Recent experiments by us with
incomplete and complete arterial injections of low
viscosity resin have confirmed that in the rat cerebral
hypophysis, the resin perfused into the median eminence flows into the anterior lobe (Murakami et al.,
1993a).
It is noteworthy that the stomach, especially its
mucosa, is perfused with the casting medium as
promptly as the kidney. Also noteworthy is that the
pancreatic islets are replicated as promptly as the
kidney glomeruli. These findings indicate that the
stomach and the pancreatic islets are the organs which
are most rapidly and richly supplied with blood.
505
This paper supplements our previous study (Miyake
et al., 1992), and demonstrates that in the rat pancreas,
the vascular plexuses of the islets are filled with the
casting medium more promptly than those of the
lobules and ducts, and that in the lobules containing
the islets, the medium flows more rapidly through the
insulo-acinar portal routes than the lobular routes via
the acinar arteries. It also shows that even in the
intralobular islets with well-developed insulo-venous
efferent vessels, the casting medium flows in the first
instance into the portal vessels. These facts prove that
the arterial blood in the rat pancreas preferentially
flows through the insulo-acinar portal routes. Moreover, we show that neither the insulo-venous routes nor
the lobular and ductal routes interfere with the insuloacinar portal routes, which convey high concentration
of insular hormones to the exocrine acini (Fujita, 1973;
Fujita and Murakami, 1973). Our recent study of
thoroughly and partially replicated specimens has
shown that even in the human pancreas in which most
of the islets are intralobular and have few collateral
venous (insulo-venous) drainage (Murakami et al.,
1992), the arterially injected resin preferentially flows
through the insulo-acinar portal routes (Murakami et
al., 1994).
Prompt filling with resin of the cortical capillaries of
the islets and later filling of their deep capillaries
support our previous findings that in the rat islets, the
blood mainly flows from the A–D cell mantle to the B
cell core (Fujita, 1973; Ohtani et al., 1986), and
strengthen our idea that in the rat islets, the B cells’
release of insulin is regulated by glucagon and somatostatin from A and D cells more effectively than otherwise by this intra-insular microcirculatory design from
the superficial to deep layers (Fujita and Murakami,
1973).
Late filling of the ductal capillaries with resin suggests that the ductal plexuses receive a small amount of
blood to reabsorb water from the pancreatic juice
(Murakami and Fujita, 1992).
Microcirculation patterns of blood have been studied
by intravital light microscopy of living tissues or organs
which are sometimes injected with India ink, fluorescent dyes, microspheres or other substances (Bunnag et
al., 1963; Frazor and Henderson, 1980; Lifson et al.,
1980; McCuskey and Chapman, 1969; Ohtani, 1983;
Ohtani et al., 1986). Although these methods are useful
for direct observation of blood flow, their use is limited;
deep and wide areas are hardly observed by the intravital microscopy. Furthermore, the images from the intravital methods are not always clear because of the
limited resolution and shallow focus of the light microscope (Ohtani, 1983; Ohtani et al., 1986). This shortcoming is supplemented by the incomplete casting/scanning technique introduced in the present study.
Retrograde flow of the injected resin in the veins
seems to be a major problem of this technique. To
overcome this problem, complete opening of the efferent
vessels of the organs to be examined is useful. The
incompletely injected or cast specimens are easily deformable so that they should be freeze-dried.
506
T. MURAKAMI ET AL.
Fig. 11. Incompletely replicated rat pancreas (8.0 ml resin injection). The islets (arrowheads) and their insulo-venous and insuloacinar portal systems are reproduced together with their connecting
interlobular veins (IV), whereas the lobular capillaries directly sup-
plied by the acinar branches of the lobular artery are not replicated.
Arrows indicate the retrograde flow in the veins. IA, interlobular
artery. Bar, 500 µm.
MICROCIRCULATORY PATTERNS IN THE RAT PANCREAS
507
Fig. 12. An insulo-lobular complex replicated by 8.0 ml resin
injection (closer view of a part of Figure 11). The lobular capillary
plexus (EL) is sufficiently reproduced together with the insulo-acinar
portal vessels (arrowheads) which arise from the islet (II). The
collateral insulo-venous efferent vessels (iv) are also sufficiently
reproduced. The capillary plexus of the secretory duct is partially
replicated (SD). IA, interlobular artery; IV, interlobular vein. Bar,
100 µm.
REFERENCES
Miyake, T., Murakami, T., and Ohtsuka, A. (1992) Incomplete vascular
casting for a scanning electron microscope study of the microcirculatory patterns in the rat pancreas. Arch. Histol. Cytol., 55:397–406.
Murakami, T. (1971) Application of the scanning electron microscope
to the study of the fine distribution of the blood vessels. Arch. Histol.
Jpn., 32:445–454.
Murakami, T. (1972) Vascular arrangement of the rat renal glomerulus. A scanning electron microscope study of corrosion casts. Arch.
Histol. Jpn., 34:87–107.
Murakami, T. and Fujita, T. (1992) Microcirculation of the rat pancreas, with special reference to the insulo-acinar portal and insulovenous drainage systems: A further scanning electron microscope
study of corrosion casts. Arch. Histol. Cytol., 55:453–476.
Murakami, T., Unehira, M., Kawakami, H., and Kubotsu, A. (1973)
Osmium impregnation of methyl methacrylate vascular casts for
scanning electron microscopy. Arch. Histol. Jpn., 36:119–124.
Murakami, T., Ohtani, O., Ohtsuka, A., and Kikuta, A. (1983) Injection
replication and scanning electron microscopy of blood vessels. In:
Biomedical Research Applications of Scanning Electron Microscopy,
vol. 3. G.M. Hodges and K.E. Carr, eds. Academic, London, pp. 1–30.
Murakami, T., Kikuta, A., Taguchi, T., Ohtsuka, A., and Ohtani, O.
Bonner-Weir, A. and Orci, L. (1982) New perspective on the microvasculature of the islets of Langerhans in the rat. Diabetes, 31:883–889.
Booz, K.H. (1964) Zur Morphologie und funktionellen Bedeutung einer
Spiralklappe in der V. portae der Nagetiere. Anat. Anz., 115:141–147.
Bunnag, S.C., Bunnag, S., and Warner, N.E. (1963) Microcirculation in
the islets of Langerhans of the mouse. Anat. Rec., 146:117–123.
Frazor, P.A. and Henderson, J.R. (1980) The arrangement of endocrine
and exocrine pancreatic microcirculation observed in the living
rabbit. Q. J. Exp. Physiol., 65:151–158.
Fujita, T. (1973) Insulo-acinar portal system in the horse pancreas.
Arch. Histol. Jpn., 35:161–171.
Fujita, T. and Murakami, T. (1973) Microcirculation of monkey
pancreas with special reference to the insulo-acinar portal system. A
scanning electron microscope study of vascular casts. Arch. Histol.
Jpn., 35:255–263.
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. (1969) Microscopy of the living
pancreas in situ. Am. J. Anat., 126:395–408.
508
T. MURAKAMI ET AL.
(1987) Blood vascular architecture of the rat cerebral hypophysis
and hypothalamus. A dissection/scanning electron microscopy of
vascular casts. Arch. Histol. Cytol., 50:133–176.
Murakami, T., Fujita, T., Taguchi, T., Nonaka, Y., and Orita, K. (1992)
The blood vascular bed of the human pancreas, with special
reference to the insulo-acinar portal system. Scanning electron
microscopy of vascular casts. Arch. Histol. Cytol., 55:381–395.
Murakami, T., Miyake, T., Ohtsuka, A., Kikuta, A., and Taguchi, T.
(1993a) Microcirculatory patterns in adult rat cerebral hypophysis:
A scanning electron microscope study of replicated specimens. Arch.
Histol. Cytol., 56:243–260.
Murakami, T., Fujita, T., Miyake, T., Ohtsuka, A., Taguchi, T., and
Kikuta, A. (1993b) The insulo-acinar and insulo-venous drainage
systems in the pancreas of the mouse, dog, monkey and certain
other animals: A scanning electron microscopic study of corrosion
casts. Arch. Histol. Cytol., 56:127–147.
Murakami, T., Fujita, T., Tanaka, T., Tsubouchi, M., Tsubouchi, Y.,
Taguchi, T., Ohtsuka, A., and Kikuta, A. (1994) Microcirculatory
patterns in human pancreas: Supplementary observations of vascular casts by scanning electron microscopy. Arch. Histol. Cytol.,
57:9–16.
Ohtani, O. (1983) Microcirculation of the pancreas: A correlative study
of intravital microscopy with scanning electron microscopy of vascular corrosion casts. Arch. Histol. Jpn., 46:315–325.
Ohtani, O., Ushiki, T., Kanazawa, H., and Fujita, T. (1986) Microcirculation of the pancreas in the rat and rabbit with special reference to
the insulo-acinar portal system and emissary vein of the islet. Arch.
Histol. Jpn., 49:45–60.
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