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Electron microscopic identification of different cell types in the islets of langerhans of the guinea pig rat rabbit and dog.

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ELECTRON MICROSCOPIC IDENTIFICATION
O F DIFFERENT CELL TYPES I N T H E ISLETS O F
LANCERHANS O F T H E GUINEA PIG,
RAT, RABBIT AND DOG1
PAUL E. LACY'
Drpurlment of Anatomy, Washington University School
of Metlicinc., St. Louis, .Miasowi
SEVEN FIGURES
The object of this investigation was t o establish an electron
microscopic identification of the different types of cells present in the islets of Langerhans. This identification of the
islet cells in different species of animals was considered
essential to the application of electron microscopy to thc
study of experirricntal diahctes. Dalton ( ,51) and Robertson
( '54) have recorded observations on the ultrastructure of
the islets of Langcrhans in the course of studies on the mouse
pancreas but a specific idcntification of the cell typcs was not
madc. In the prcsent study, the identification of the cell
types was established by comparing the same cells observed
in a low power electron micrograph and in a contiguous thick
section which was examined by light microscopy.
MATERTAL AND METHODS
Five guinea pigs, 5 rats, 4 dogs and 4 rabbits were uscd.
The animals were killed by a blow to the head o r by intravenous nembutal. A portion of thc pancreas was removed
rapidly, cut into small (1mm) pieces and placed in a small
This investigation W ~ H aided in part by grant RG-3784 from the National
Institutes of IIealth, U. S . Puhlic Health Service.
* Postdoctorate Research Fellow of the National Institutes of Health, IT. 5.
Puhlic TTealth Service.
255
256
P A U L E. LACY
amount of osmic acid- dichromate fixative buffered to pH
7.2 or 7.6 (Dalton, '55). The tissues were fixed at room temperature for one to two hours, then rinsed with distilled water
and dehydrated through a graded series of ethanol solutions.
The tissues were then transferred to a half and half mixture
of absolute ethanol and methacrylate followed by two changes
of methacrylate. The methacrylate was composed of 8 p a r t s
n-butyl methacrylate to one p a r t methyl methacrylate. The
tissues were embedded in partially polymerized methacrylate
which was further polymerized at 45°C. for 24 hours. Benzoyl
peroxide was used to catalyze the reaction. Sections were
cut with glass knives on a Servall microtome. Thick sections
mere examined by phase microscopy in order to localize the
islets within the blocks of pancreas. A thin section was obtained for electron microscopy and a contiguous thick section
was cut f o r staining. The thin sections were floated onto
acetone-water, lifted on a collodion-coated, copper mesh grid
and examined i n a n RCA electron microscope (EMU 2C).
A low power objective pole piece was used in order to obtain
initial magnifications of approximately 575 diameters. The
contiguous sections were floated onto glass slides arid affixed
to the slide by warming. The methacrylate was removed by
extracting the sections with benzene o r toluene for 30 to 45
minutes. The sections were oxidized in 1% periodic acid
for 5 to 10 minutes. Aldehyde fuchsin (Gomori, '50) was
used to stain the beta cells and a light green-orange G mixture
was used as a counterstain.
Photomicrographs of the stained sections were made at
approximately the same initial magnifications as the low power
electron micrographs and both were enlarged photographically a s desired. The same islet cells could then be observed
and compared in the electi.on micrographs and the photomicrographs.
OBSERVATIONS
Gwineu pig. The compact arrangement of the islet cells
and the small size of their secretion granules made it possible
ELECTRON MICROSCOPY OF THE ISLETS
257
to distinguish easily the islets of Langerhans from the surrounding acinar cells by low power observation with the
electron microscope. The islct cells were rounded o r polygonal in shape and frequently revealed protoplasmic processes
extending between adjacent cells. These intervening, protoplasmic processes were most frequently observed along the
capillaries. Three morphological types of islet cells were
observed in the low power electron micrographs. These cells
were identified as alpha, beta and C cells by examining the
same cells in a contiguous, stained, thick section (figs. 1 and
2 ) . In the low power electron micrographs, the cytoplasm
of the alpha cells appcarcd pale and contained numerous,
dense, round granules. Their nuclei were pale and were usually
ovoid in shape. The alpha cells were scattered singly or in clusters along the capillaries. The beta cells appeared darker and
contained granules which were less dense and more irregularly
shaped than those of the alpha cells. Thc darker appearance of
the beta cells was apparently due to a greater amount of ergastoplam in their cytoplasm (fig. 6). The nuclei of the beta
cells were dense and wcre generally rounded in shape (fig. 2).
This nuclear differencc between the alpha and beta cells of
the guinea pig is similar to that which has been observed by
light microscopy (Bensley, '11). The C cells were characterized by a very pale cytoplasm that appeared devoid of
distinct granules. They werc usually observed as individual
cells Scattered throughout the islets. The nuclei of all three
types of cells contained one or more nucleoli.
Rabbit. Two different types of islet cells were evident in
the low power electron micrographs as illustrated in figure 3.
By examining thc same ~ ~ 1in
1 sa stained section, their identity
was established as alpha and beta cells. The cytoplasm of
the alpha cells was packed with dcnse, round granules whereas
the cytoplasm of the beta cells appeared pale and the beta
granules could not be distinguished from mitochondria in the
low power electron micrographs. On higher magnification,
the beta granules were observed but they were less dense
and smaller than the granules of the alpha cells. The beta
258
PAUL E. LACY
cells were also characterized by a light zone which partially
or completely surrounded theifi nuclei (fig. 3 ) . This light zone
was not observed in the beta cells of the guinea pig, dog or
rat. The alpha cells were usually found at the periphery
of the islets which is a characteristic position of these cells
in the rabbit pancreas (Thomas, '37). The nuclei of both
types of cells were round or ovoid in shape and there were
no apparent diff ercnccs in nuclear structure between the
alpha and beta cells.
Dog. The islet cells were somewhat columnar in shape and
showed a tendency t o palisade around the capillaries. The
nuclei of the islet cells revealed a variety of shapes appearing elongated, oval or round and the nuclear coiitours were
markedly irregular. Alpha and beta cells were identified and
distinguished morphologically in the low power electron micrographs (fig. 4). The major electron microscopic difference
between the two types of cells was that the alpha cell granules
were dense and rounded in shape whereas the majority of the
beta granules were less dense and were rod-shaped. These
rod-shaped granules were surrounded by a clear space in
the beta cell cytoplasm (fig. 7 ) . A few rounded granules
were also present in the beta cells. This rod-shaped configuration of the beta granules was observed in all of the dogs
examined and was not observed in the beta cells of the guinea
pig, rabbit or rat. Occasional cells were observed which
appeared degranulated. There were no distinguishing nuclear
differences between the alpha and beta cells.
Rat. In the low power electron micrographs, two types of
cells were observed in the islets of Langerhans as illustrated
in figure 5. The staining of the contiguous thick sections was
unsatisfactory so that it was not possible to make a definite
identification of the alpha and beta cells on the basis of the
stained sections. One of the cell types was characterized by
a pale cytoplasm containing dense, round granules and a pale
nucleus. These cells were observed predominantly at the
periphery of the islets and comprised only a small portion
of the total islet cell population. Since it has been reported
ELECTKOS MICKOSCOPY O F THE ISLETS
259
that in the normal rat the alpha cells occupy the periphery
of the islet and comprise the minority of the islet cells (Gomori,
'39), this first type was identified as the alpha cell. The
second type, believed to be the beta cell, had a dark cytoplasm
containing dense, round granules and a dark nucleus. The
darker appearance of the cytoplasm of the beta cell was
apparently due to a greater content of ergastoplasm. This
difference between the two types of cells was similar to the
difference observed between the alpha and beta cells of the
guinea pig (fig. 6). The nuclei of both types of cells were oval
or rounded in shape.
DISCUSSION
The electron microscopic identification of the islet cells
was facilitated by using low magnifications since several islet
cells could be examined and compared in the same electron
micrograph and these cells could be easily matched with the
same cells observed in a photomicrograph of a contiguous
stained section. The staining of the thick sections with
Goniori 's aldehyde fuchsin method (Gomori, '50) produced
a clear distinction between alpha and beta cells in the islets
of the rabbit, dog and guinea pig. The most intense staining
of the beta cells was observed in the guinea pig islets. I n the
rat pancreas, a differentiation of the alpha and beta cells
was not obtained in the stained sections.
The differentiation of alpha and beta cells in the islets of
Langerhans has been made chiefly by selective stains such
as aldehyde fuchsin (Gomori, '50) or chrome alum hematoxylin (Gomori, '39) to demonstrate beta cells or silver stains
(Ferner, '38 ; Damon and Barnett, '44) to demonstrate alpha
cells. By light microscopy, distinct morphological differences
observed between the two types of cells are relatively few
except f o r the varying position of the alpha cells in the islets
of different species (Korp and LeCompte, ' 5 5 ) . Electron
microscopic study of the islets revealed morphological differences in the ultrastructure of the two cell-types so that a
differentiation of the cells could be made on the basis of the
260
P A U L E. LACY
size, number, shape and dcrisity of the granules, the arnount
of ergastoplasm and differences in nuclear density. The
cytological features of the beta cells were distinctly different
in each of the various species of animals that were examined.
The beta granules were irregularly shaped in the islets of
the guinea pig, bar-shaped in the islets of the dog, small,
round and indistinct in the islets of the rabbit and round and
relatively dense in the islets of the rat. A light zone which
partially or completely surrounded the nuclei of the beta
cells was observed only in the islets of the rabbit. The alpha
cclls were more uniform in structure in the various animals
and the granules of the alpha cells appeared denser than
the beta cell granules. Further studies of these cytological
differences of the islet cells are being made at higher magnifications with the electron microscope
SUMMARY
The islets of Langerhans of the adult guinea pig, rabbit,
dog and rat were studied with the electron microscope at
low magnifications.
Electron microscopic identification of the alpha and beta
cells was made in each of the different species of animals.
The identification of the islet cells was verified by comparing
the same cells observed in a low power electron micrograph
and in a photomicrograph of a contiguous stained section.
A marked variation in the ultrastructure of the beta cells was
found in each of the different species of animals that were
studied.
ACIK N0WLEI)GMENT
I wish to thank Dr. E. W. Dempsey for his advice and
criticism during the course of this protjcct.
J J T E R A T U R E CITED
BENSLEY,
R. R. 1911 Studies on the pancreas of the guinea pig. Am. J. Anat.,
12: 297-388.
DALTON, A. J. 1951 Electron inicrography of epithelial cclls of the gastrointcstirial tract and pancreas. Am. J. h a t . , 89 : 109-133.
ELECTKON MICBOSCOPY O F THE ISLETS
261
DALTON,A. J. 1955 A chrome-osmiuni fi vative f o r elcctroii iiiicroscopy. Anat.
Rec., 221: 281.
DAWSOX,A. B., AND J. RARNETT 1944 Rodian’s protargol method applied t o
other than neurological preparations. Stain Technol., 29 : 115-118.
FEnNER, H. 1938 Uber die Entwicklung der Langerhansschen Inseln nach der
Geburt und die Bedeutuiig dcr versilherbaren Zcllen im Pancreas dcs
Menschen. Ztsch. f . mikr.-anat. Forsch., 4 4 : 451-488.
GOMORI, G. 1939 Studies on the cells of the pancreatic islets. Anat. Rec., 7 4 :
439-459.
1939 A differential staiii f o r cell types in t h e pancreatic islets.
Am. J. Path., 15: 497-499.
1950 Aldehyde-fuelisin: A n e w stain for elastic tissue. Am. J. Clin.
Path., ZU: 665-666.
KonP, W., AND P. M. LECOMPTE195.5 The nature and function of the alpha
cells of t h e pancreas. Diabetes, 4 : 347-366.
ROBEKTSOS,J. S. 1954 A morphological study with the electron microscope of
sections of the normal mouse pancrras. Australian J. Erper. Biol. and
Med. Sc., 32: 229-234.
THOMAS,
T. B. 1937 Cellular cornpoileiits of the mammalian islets of Langerhans.
Am. J. Anat., 6 8 : 31-57.
PLATE 1
EXPLANATION OF FIGURES
1
Photomicrograph of a guinea pig islet stitinetl with Goniori 's adlchydc fuchsiu
and an orange G-light grerii mixture. The beta cells (B) appear black, alpha
cells ( A ) grey and C cells (C) clear or p l e grey i n the photograph. X 1,300.
2
Electroii iiiicrogmph of a coiitiyuous wetion of the guinea pig islet shown
in figure 1. The arrowb point t o the same cells t h a t were sliown in figure 1.
The alpli eells ( A ) have a pale, background cytoplasm, pale, ovoid nuclei aud
contain numerous, densc? granules. The beta cells (B) have darker, round
iiuclri, darker background cytoplasm and less dense granules. The C cells
( C ) ;ippe:tr pale with 110 distinct granules. X 2,300.
262
263
PLATE 2
EXPLANATION O F FIGURES
3
Electron micrograph of a portion of an island of Langerhans from a rabbit.
The alpha cells ( A ) contain numerous, dense granules. The beta cells (B)
have a clear zone ( C ) partially or completely surrounding the nuclei and the
brta granules can not be cloarly distinguished a t this magnification. The
large zymogen granules ( Z ) of the acinar cells are shown on the left. X 2,300.
4 Electron micrograph of a portion of an island of Langerhans from the dog.
The alpha cells ( A ) contain dcnse, round granules. The majority of tho
granules in the beta cells (B) appear rod-shaped and are surrounded by a
clear area. The nuclei of both types of cells show an irregular contour. Acinar
cells containing zymogcn granules ( Z ) me shown on both sides of the islct.
X 2,300.
5
Electron micrograph of a portion of an island of Langerhans from the rat.
The alpha cells (A) have a pale, background cytoplasm, pale nuclei and dense,
round granules. The beta cells (B) have a darker Cytoplasm, darker nuclei
and round and less dense granules. The alpha cells were a t the periphery of
the islet adjacent t o the aciiiar cells containing eymogen granules (2).
X 2,300.
264
E I J E O T R O X X I C R O S C O P P O F THE lYLETS
PAUL E. LACY
PL.4TE 2
PLATE 3
EXPLANATION OF FIGURES
0
Electron micrograph of an alpha (A) and beta cell (B) from a guinea pig
islet. The beta cell (B) has a darker nucleus ( N ) and contains a greater
ainount of ergastoplasm ( E R ) than the alpha cell ( A ) . The granules (G)
of the beta cell are slightly larger, less dense and more irregularly shaped than
the granules ( G ) of the alpha cell. X 13,500.
7
Elcctron micrograph of a portion of a beta cell from a dog. Thr majority
of the beta granules (G) are rod-shaped and are surrounded by a clear apace.
Ergastoplasm (ER) and several mitochondria ( M ) are also present. X 22,500.
266
RI,EC,TRON 3TICROSCOPY O F THE ISLETS
I’IUL, E. LACY
PLATE 3
267
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