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Microcirculation in the islets of langerhans of the mouse.

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Microcirculation in the Islets of Langerhans
of the Mouse'
Department of Pathology, The University of Chicago, Chicago, Illinois
A technique for the study of microcirculation in living islets, using
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."
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
The tail veins were used for intravenous
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.
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
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-
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).
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-
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
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
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.
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-
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).
Anderson, E., and J. A. Long 1947 The effect
of hyperglycemia on insulin secretion as determined with the isolated rat pancreas in a
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Banting, F. G., and S. Gairns 1924 Factors influencing the production of insulin. Amer. J.
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circulation. Proc. SOC. Exp. Biol. Med., 27:
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1958 Studies on the vascular system of the
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Freedman, P., R . Moulton, M. L. Rosenheim, A.
G. Spencer and D. A. Willoughby 1958
Phaeochromocytoma, diabetes and glycosuria.
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Gomori, G., N. B. Friedman and D. W. Caldwell
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rat with reference to intermittent corpuscular
flow i n individual capillaries. Quart. J. Exp.
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