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Isolated pancreatic islets of the ratAn immunohistochemical and morphometric study.

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THE ANATOMICAL RECORD 237:489-497 (1993)
Isolated Pancreatic Islets of the Rat:
An lmmunohistochemical and Morphometric Study
MOSTAFA M. EL-NAGGAR, AHMED A. ELAYAT, M. SALLEH M. ARDAWI, AND
MOHAMMAD TAHIR
Departments of Anatomy (M.M.E.-N., A.A.E., M.T.) and Clinical Biochemistry (M.S.M.A.),
Faculty of Medicine and Allied Sciences, King Abdulaziz University, Jeddah,
Kingdom of Saudi Arabia
ABSTRACT
Although there is a recent increase in the use of the isolated
pancreatic islets of the rat in the transplantation and functional studies,
there has been no detailed quantitative assessment on the size and cellular
constituents of islets after the isolation procedure. The present work was
undertaken to study the size classes of the isolated islets and the morphometry of their cellular populations.
Islets of the rat pancreas were isolated by using the intraductal collagenase digestion technique, the most commonly used procedure for the isolation of pancreatic islets. Different endocrine cells of the isolated islets
were stained by immunoperoxidase staining techniques. The distribution
of the cellular constituents of the isolated islets was similar to that of the
intact islets of the normal pancreas; A, D, and PP cells were peripherally
arranged around the centrally located B cells. However, morphometric
quantitative study showed that the percent volume and percent number of
A, D, and PP cells of the isolated islets were lower than those of the corresponding intact ones. Further, the mean true diameter of the isolated islets
was lower than that of the intact ones. These data indicate loss of islet cells
during the process of isolation. Most of the lost cells were from the periphery of islets. This may provide an explanation for the incomplete metabolic
control and recurrence of hyperglycemia encountered after isolated islet
transplantation in the treatment of diabetes mellitus. It seems that further
refinements of the isolation techniques are necessary to obtain islet tissue
with total cellular integrity, before a complete success in transplantation
could be achieved. o 1993 Wiley-Liss, Inc.
Key words: Immunohistochemistry, Isolated islets, Morphometry, Pancreas
Moskalewski (1965) was the first to isolate islets
from the pancreas of the guinea pig using a n enzymatic
digestion technique. Lacy and Kostianovsky (1967)
modified the technique and used the density gradients
to purify isolated rat islets. Further improvement of
the technique was made by Sutton et al. (1986) who
used the method of intraductal injection of collagenase.
This technique facilitated harvesting large number of
islets for use in transplantation procedures to treat diabetes (Sutherland et al., 1980; McEvoy and Leung,
1983; Toledo-Pereyra et al., 1984; Alejandro et al.,
1986; Dibelius et al., 1986; Hesse et al., 1986; Lake et
al., 1988; Tze and Tai, 1988; Huxlin et al., 1990). The
isolated islets were also used to evaluate their ability
to secrete insulin in vitro in response to glucose load
(Lacy et al., 1968; Slavin e t al., 1977; Halban et al.,
1986; Eizirik et al., 1989; Grodsky, 1989).
The process of isolation inevitably leads to mechanical and enzymatic trauma to the islets themselves
(Moskalewski, 1965;Vance et al., 1968; Buchanan and
Mawhinney, 1973; Slavin et al., 1977) especially to
0 1993 WILEY-LISS, INC.
their peripheral part, which is rich in A, D, and pancreatic polypeptide (PP)cells. The central core of islets,
which is formed of insulin-producing B cells, was kept
almost intact. These changes in the peripheral part of
isolated islets presumably affect the hormonal secretory function of B cells. There is growing evidence that
glucose-induced insulin release is dependent not only
on the integrity and number of the insulin-containing
B cells, but also on their interactions with the neighboring B and non-B cells (Pipeleers et al., 1982). In
these studies, however, the effect of the isolation procedure on the different cellular populations and the
Received March 9, 1993; accepted July 26, 1993.
Address reprint requests to Dr. Mostafa M. El-Naggar, Department
of Anatomy, Faculty of Medicine and Allied Sciences, King Abdulaziz
University, P.O. Box 9029, Jeddah 21413, Kingdom of Saudi Arabia.
490
M.M. EL-NAGGAR ET AL.
size of the pancreatic islets, was not quantitatively documented.
In the present work the main objective was to study
immunohistochemically the distribution of the cellular
constituents of the functionally viable isolated pancreatic islets of the rat together with morphometric evaluation of the different cell types and the size classes of
the isolated islets. Intact islets of normal pancreases
were similarly studied and were used for comparison.
MATERIALS AND METHODS
Isolation of the Pancreatic lslets
The pancreatic islets were isolated from adult male
Lewis rats using the ductal perfusion method of Sutton
et al. (1986) which is based on the original collagenase
digestion technique of Lacy and Kostianovsky (1967).
The rats were anaesthetized with ether inhalation, and
the common bile duct was cannulated with a fine polyethylene tube (PE-50) immediately after incision of the
abdomen. The duct was then ligated near the duodenum, and the rat was bled to death. The pancreas was
infused with 7 ml of cold, freshly prepared, collagenase
[Sigma type V (lot 100H6851)I in medium M199. The
pancreas was quickly excised and incubated a t 37°C.
Different concentrations of collagenase (1,2, and 3 mg/
ml) and different digestion times (16,18,20,22,24,26,
and 28 min) were used to obtain the optimal digestion
conditions for this batch of collagenase. The action of
collagenase was stopped by adding cold medium M199.
The digested pancreatic tissue was disrupted by aspiration through a 14-gauge cannula. The aspirate was
filtered through a Nytex filter (mesh size 1,000 pm),
collected into a centrifuge tube and the volume was
adjusted with cold medium M199 and centrifuged a t
400g for 10 min at 4°C. The supernatant was decanted
and the pellet was resuspended in the bottom layer of a
discontinuous Ficoll gradient with a density of 1.095.
Other Ficoll gradient layers with densities of 1.085,
1.075, and 1.045 were carefully layered on top of the
bottom layer respectively, and the tube was then centrifuged at 8OOg for 20 rnin a t 4°C. The islets usually
collect at the first and the second interface (between
the layers with densities of 1.045, 1.075, and 1.085,
respectively). The islets were aspirated and placed into
another centrifuge tube and washed twice by using cold
medium M199; each time these were centrifuged a t
400g for 10 min a t 4°C. The pellet of the islets was
resuspended in the medium and transferred to a petri
dish. The islets were then further purified by hand
picking of the non-islet tissue with the help of a binocular dissecting microscope with a black background,
illuminated by two horizontal beams of fiberoptic light.
The whole process was carried out under a laminar
flow hood to avoid contamination of the isolated islets.
The islets isolated from each rat pancreas were
counted under the dissecting microscope immediately
after purification and the counting was confirmed under phase contrast microscope by a second observer.
Using a calibrated grid attached to the phase contrast
microscope the purity of the islet preparation was evaluated. The purity was calculated as the percent number of the intersections which overlie islets to the number of the intersections which overlie islet and non-islet
tissues.
Functional Evaluation
Aliquots from the islet preparations which were isolated with different concentrations of collagenase and
with different digestion times were functionally evaluated by measuring their insulin and C-peptide contents. Insulin was extracted from the isolated islets by
acidified ethanol and quantitated by radioimmunoassay procedure using Coat-A-Count kit. C-peptide concentrations were measured by double antibody radioimmunoassay technique using commercially available
kit (Diagnostic Products Corporation, Los Angeles,
CA). Radioactivity was determined using Beckman
gamma counter Model 5500 for 1min. The results were
expressed as units of insulin or C-peptide per g of protein. The protein content of islet preparations was determined by the procedure of Lowry et al. (1951).
The islets isolated with the optimal digestion conditions (22 min incubation time and collagenase concentration of 2 mg/ml) were further functionally evaluated
by determining their adenine nucleotide contents. ATP
concentration was measured by the method of Lamprecht and Trautschold (1974). ADP and AMP concentrations were measured by the method of Jaworek et al.
(1974). Samples of freshly prepared isolated islets were
incubated in 1 ml of medium M199 containing 5.5
mmol/liter glucose. Incubations were terminated by
the addition of 200 p1 of HC10, (25%, w/v) to the incubation medium and cooling the mixture to 0°C a t 0,30,
and 60 min of incubation time. The precipitated proteins were removed by centrifugation a t 13,500g for 2
min. The supernatant was neutralized with KOH containing 0.5 M-triethanolamine, and the KClO, was removed by centrifugation at 13,500g for 3 min. Adenine
nucleotide concentrations were measured in neutralized extracts of the islets with a Beckman DU-6 recording spectrophotometer. The Concentrations of the nucleotides were expressed as nmol/mg of protein.
Adenine nucleotide concentrations were also measured
in freeze-clamped tissues of intact pancreatic islets using the procedure described by Ardawi and Newsholme
(1985).
The ability of the isolated islets to secrete insulin
was evaluated at the optimal digestion conditions. The
isolated islets were incubated in a 0.5 ml syringe barrel
containing medium M199 with 1%(w/v) bovine serum
albumin (fraction v). The incubation chambers were
gassed with 02/C02(1911) and incubated a t 37°C in a
shaking water bath a t 50-60 oscillations/min. An
equilibration period of 15 min was allowed and then
the islets were challenged twice for 30 min each with
medium M199 supplemented with 15 and then 30
mmoYliter glucose, separated by a 60 min rest phase
with medium M199 only. After a further rest phase of
60 min, a final challenge of 15 mmol/liter glucose together with 15 mmol/liter theophylline were used to
elicit a maximal response from the islets (see Henquin
and Meissner, 1984).The insulin release in response to
challenge was determined and results were expressed
as units of insulin released per unit time per mg of
protein.
lrnrnunohistochernical Staining
Islet specimens isolated from eight rats, with the optimal digestion conditions described in the present
491
ISOLATED PANCREATIC ISLETS OF THE RAT
work, were fixed in buffered neutral formalin and embedded in paraffin with 57°C melting point. Five micron serial sections were cut from each specimen, and
groups containing 6-8 sections were selected a t 70 p-m
intervals from each other. Sections from each group
were stained immunohistochemically for insulin, glucagon, somatostatin and PP-containing cells. Paraffin
blocks were also prepared from different areas of the
pancreases of 6 rats, matched for age, sex, and weight
to those of the donor rats used for isolation of the islets.
Sections were cut and stained in a similar way to that
used for the isolated islets, and were used for comparison.
To localize the insulin producing B cells, the indirect
immunohistochemical technique was used (Sternberger, 1979). The primary antibody used was guinea
pig anti-swine insulin serum (lot 030), diluted in phosphate-buffered saline (PBS) with 1%normal rabbit serum. Different dilutions were used to obtain the optimal one; this was found to be 1500 which gave the best
staining with the least background and was used for
staining the sections thereafter. The secondary antibody used was rabbit anti-guinea pig immunoglobulin
conjugated with peroxidase (dilution 1:200).
The avidin-biotin complex (ABC)technique was used
to localize glucagon, somatostatin and PP-producing
cells as described previously (Hsu et al., 1981). The
primary antibodies used were rabbit anti-porcine glucagon serum (lot O l l ) , rabbit anti-human somatostatin
serum (lot 050e), and rabbit anti-human PP serum (lot
109e). They were tried in dilutions ranging from
1:10,000 to 1:lOO in PBS with 1%normal swine serum.
It was found that the optimal dilutions of the antiglucagon, anti-somatostatin and anti-PP sera were
15,000, 1500, and 15,000, respectively. These dilutions were used for all the staining procedures thereafter. The secondary antibody used was biotinylated
swine anti-rabbit immunoglobulin (dilution 1:200).
Sera and antisera were obtained from Dako Corporation (Carpenteria, CA). Sections were incubated in the
primary antibodies for overnight in a humidity chamber, a t 4°C. The chromogen substrate used was 3,3-Diaminobenzidine tetrahydrochloride (Sigma, St. Louis,
MO) and the sections were counterstained with Harris’
hematoxylin to facilitate the nuclear identification.
Specificity control for the immunohistochemical
staining included omitting the antibody sera, which
were replaced by non-immune sera, and absorption of
the primary antisera with the purified antigens.
method of Weibel (1963). With a calibrated grid inserted into the ocular lens of the microscope, the total
number of the intersections over the cytoplasm and the
nucleus of the specified cell type (Pc) as well as over the
whole islet (Pi)were determined. The percent ratio of
the number of intersections of the specific cell type per
total islet intersections Ppc = (Pc/Pi) x 100 was expressed as the volume density of this cell type for the
islet quantitated (Vvc = Ppc).
The numerical percent of each specific cell type (NNc)
per total islet cells was calculated using the nucleus as
the counting base. The nuclei of the stained and unstained cells per islet profile were counted. The ratio of
specific cell nuclei to the total islet nuclei, was expressed as the percent number of that specific cell per
islet cells NNC= (NcINi) x 100. Calculations for B, A,
D, and PP cells were done in adjacent or semi-adjacent
sections stained specifically for these types of cells.
The profile diameters (d) of the islets were calculated
where a and b are the
from the equation d = 2
major and major a t right angle semi-axis, respectively
(Williams, 1977).The size-frequencydistribution of the
profile diameters of the isolated and the intact islets
were plotted. The mean axial ratio of the profiles was
calculated. Assuming that the islets are spheroid structures, the formula of Fullman (Williams, 1977), for the
ungrouped profile range ofsizes, was used to calculate
the mean true diameter (D) for the isolated and the
intact pancreatic islets.
a,
N
7F
D=X
2
l/dl
+ lid2 . . . . 1/&
where N represents the total profiles measured and d l ,
d2, . . . . dN represent the profile diameters.
The results of the morphometric study were presented as the arithmetic mean 2 standard error of
mean. Student’s t-test for non-paired observations was
used for statistical evaluation of the data.
RESULTS
The freshly prepared isolated islets are recognized
under the dissecting microscope by their characteristic
well-defined, rounded or oval shape, showing high
opacity and milky white color. They are also recognized
under the phase-contrast microscope, as well-defined
rounded or oval bodies with a faint green tinge, in contrast to the acinar tissue which appeared irregular in
shape and showed a lighter tinge. The series of experiments which were performed to obtain the optimal
Morphometric Analysis
conditions for isolation of the islets, showed that the
Morphometric study was carried out on the immuno- highest yield of islets, (342.7 & 15.8 islets/rat pancreas)
histochemically stained sections. Twelve to twenty is- was obtained by the use of collagenase at concentration
let profiles were chosen a t random from each slide, of 2 mg/ml (Table 1) and incubation time of 22 min
with a total of 130-180 from each specimen. They were (Fig. 1).Poor yields of isolated islets were obtained
morphometrically analyzed at a magnification of with incubation periods less or greater than 22 min and
1,000 x . Sections of intact islets, taken from different with collagenase concentration of 1or 3 mg/ml. Higher
parts of 6 normal pancreases, were similarly analyzed. concentrations of insulin (318.45 t 11.3 mUlg protein)
Standard morphometric methods were used to estimate and C-peptide (176.43 2 16.44 pmol/g protein) were
the volume density (Vv) and the percent number (NN) also obtained from samples of islets isolated with colfor each cell type (WeJbel, 1963) and to calculate the lagenase a t concentration of 2 mg/ml and incubation
mean true diameter (D) of the isolated and the intact for 22 min (Fig. 2); the latter were selected as the optimal digestion conditions. The purity of the isolated
islets (Williams, 1977).
The volume densities (Vv)of B, A, D, and PP cells per islet preparations was 20-40% after separation with
islet tissue were calculated by the point counting the discontinuous Ficoll gradient. Most of the impuri-
492
M.M. EL-NAGGAR ET AL.
TABLE 1. The total number of isolated islets per rat
pancreas and the % purity of the islets obtained with
the use of 22 min incubation time and 1,2,and
3 mg/ml collagenase
Collagenase
concentration
(mglrnl)
1
2
3
No. of islets
per rat
(mean 2 SEMI
No. of
rats
6
14
6
140.7 2 11.7
342.7 2 15.8*
132.3 ? 8.6
% purity of
the islets
(mean 2 SEMI
73 2 1.8
82.6 2 1.9**
70 ? 5
*No. of the isolated islets at 2 mg vs. number at 1 or 3 mg/ml, P <
0.001.
**% uuritv a t 2 mg vs. % purity a t 1 or 3 mg, P < 0.01 and P < 0.05,
respectivdy .
400
-.s
a,
0
L
a
M
\
-
350
4
ld
i,
3;
d
a,
300
T
250
II)
..J
c
200
0
L
W
T
150
0
h
16
18
20
7
8I
22
24
300
300
250
250
200
150
r:
. A
100
50
16
7
26
:
a,
3
-E
-2
-5
4
0
p < o o 1 p<ooo1
350
4
5
400
350
400
Insulin
0C-peptide
1 11
18
20
I
22
200
a
l-
;
a
v
150
o
2
t'
24
26
1
Digestion t i m e ( m i n )
100
$.
50
I
0
a
0
28
Fig. 2. Insulin and C-peptide concentrations in aliquots of islets
isolated at different incubation times with 2 mgiml collagenase.
28
Digestion t i m e ( m i n )
Fig. 1. The number of isolated islets per rat pancreas at different
incubation times with 2 mg/ml collagenase. Values at the bottom of
each column represent the number of the rats used for isolation of the
islets.
ties consisted of pancreatic acini, in addition to small
ducts, blood vessels and lymph nodes. Handpicking of
the non-islet tissues could raise the final purity up to
82.6 t 1.9%(Table 1).
Evaluation of the functional integrity of the islets,
isolated using the optimal digestion conditions described in the present work showed that the concentrations of ATP and the total nucleotides were not significantly different from freeze-clamped intact pancreas
and the isolated islets which were either freshly isolated or incubated for 30 and 60 min (Table 2). However, ATP/AMP concentration ratio was decreased in
isolated incubated islets by 16.67% and 35.86% when
the islets were incubated for 30 to 60 min, respectively.
Repeated challenge of the isolated incubated islets
with solutions of 15 followed by 30 mmol/liter glucose
resulted in highly reproducible responses of insulin release above the baseline secretion which was 76.67 ?
16.67 pU/hr/mg protein. This response to glucose challenge was independent of the downward drifting baseline. The system functioned for 5 hr without compromising islet performance as witnessed by the maximal
response of the islets to theophylline, which led to the
release of 318.3 2 60 p.U insulin/hr/mg protein (Fig. 3).
The immunohistochemical staining of the isolated
islets showed four populations of cells which were distinct from each other not only in their specificity for
each primary antibody, but also in their respective
number and morphological distribution. The majority
of the islet cells were B cells and they reacted positively
to anti-insulin serum. The reaction of the isolated islets
to the anti-insulin serum was generally weaker than
that of the intact islets of the normal pancreas (Fig. 4,
compared with Fig. 5). In many isolated islets, particularly the large ones, the cells differed greatly in their
degree of reaction; the cells a t the periphery of the
islets were almost well granulated, whereas the cells in
the center were somewhat degranulated (Fig. 4).
The A cells reacted positively with anti-glucagon serum. They were distributed a t the periphery of the isolated islets (Fig. 6), similar to their distribution in the
intact islets (Fig. 7). A cells of the isolated islets appeared, however, less well organized than in the intact
islets. They did not form a complete mantle around the
B cells, but were rather arranged in clusters scattered
a t the periphery of the isolated islets. It was also noticed that some of the peripheral cells seemed injured
and the blood vessels on the periphery of the islets were
torn.
The D cells stained positively with anti-somatostatin
serum. They were somewhat variable in number and
were dispersed a t the periphery of the isolated islets
(Fig. 8), whereas in the intact islets they were located
either at the periphery of the islet or dispersed in an
intermediate position between the A and B cells (Fig.
9). The immunohistochemical reactivity of the D cells
of the isolated islets was weaker than that of the intact
pancreatic islets.
The PP cells stained positively with antiserum to
pancreatic polypeptide. The cells were arranged singly
or in clusters a t the periphery of the isolated islets (Fig.
10) similar to their arrangement in the intact pancre-
493
ISOLATED PANCREATIC ISLETS OF THE RAT
TABLE 2. The concentration of adenine nucleotides in the isolated pancreatic islets compared with that of the
freeze-clampedintact pancreatic tissue'
Concentration (nmol/me of Drotein)
Samnle
~
Isolated islets
Isolated islets
Isolated islets
Freeze-clamped
pancreatic tissue
Incubation
time ( m i d
0
30
60
1.34 f 0.07
1.34 t 0.09
1.28 f 0.19
0.331 f 0.064
0.315 10.029
0.379 0.027
*
0.289 I
0.043
0.341 r+ 0.037
0.422 f 0.040
1.903 I
0.231
1.986 0.099
2.083 f 0.244
*
4.74
3.95
3.04
0
1.67 t 0.25
I
0.17
0.232 t 0.057
* 0.32
7.18
?
0.39
2.30
SEM, for eight specimens of isolated islets and six specimens of freeze-clamped intact pancreatic tissue.
5 5 mmol glucose/l
15 rnmol glucose/l
30 mmol glucose/l
a
A
\
ATP/AMP
ratio
015 mmol
Qi
4
z
-2
Total adenine
nucleotides
300
h
2
AMP
350
400
G
ADP
-
'Values are presented as means
.3
ATP
glucose/l a n d
15 mmol theophylline/l
250
200
of the intact islets of the normal pancreas (Fig. 13).
This indicates loss of islet mass during the isolation
procedure which affected all sizes of the islets. The cumulative frequency distribution of the profile diameters of the isolated islets showed that their population
contain a range of sizes, justifying the use of the formula of Fullman (WiJliams, 1977) to calculate the
mean true diameters (D) for the isolated and the intact
pancreatic islets, which were found to be 104.547 and
196.319 pm, respectively.
150
v
DISCUSSION
-,s
100
-
50
3
rA
G
0
Challenge
Fig. 3.Insulin release from islets isolated with 2 mgiml collagenase
and 22 min of incubation, in response to a challenge twice with 15and
30 mmol/liter glucose, then a final challenge with 15 mmoliliter glucose together with 15 mmolfliter theophylline to elicit a maximal
response.
atic islets (Fig. 11). Whereas few isolated islets contained a large number of PP cells, most of the isolated
islets contained 1-3 PP cells or were devoid of them
(Fig. 10).
The morphometric study showed that the cellular
populations of the isolated islets were quantitatively
different from those of the intact islets. The results of
the morphometric study are summarized in Table 3.
Whereas the volume density and the percent number of
the B cells of the isolated islets were significantly
higher than those of the intact islets, the volume densities and the percent numbers of the peripherally arranged A, D, and PP cells of the isolated islets were
significantly lower than those of the intact islets (Fig.
12).
The mean profile diameter of the isolated islets was
significantly lower (P < 0.001) than that of the intact
islets (Table 3). The profile sections of the islets showed
a mean axial ratio of 1.468 in the isolated islets and of
1.345 in the intact islets, indicating that the islets
could be treated as spheroids. The size-frequency distribution of the profile diameters of the isolated islets
showed an overall shifting of all the size classes toward
smaller sizes as compared to the corresponding classes
Isolation of the islets has become a commonly used
procedure to obtain a relatively pure endocrine tissue
for transplantation experiments or for functional metabolic studies. In the present work, the islets were isolated from the pancreas of the rat by the intraductal
collagenase digestion technique of Sutton et al. (1986).
This was selected since it is the most commonly used
technique for the isolation of pancreatic islets. Our
preparations of the isolated islets proved to be functionally intact and viable. This was assessed by measuring
the adenine nucleotide concentrations in the isolated
islets, which were not significantly different from those
of the intact islets of the freeze-clampedpancreatic tissue. In addition, isolated islets which were incubated in
vitro for 30 to 60 min maintained their adenine nucleotide concentrations. Repeated challenge of the isolated incubated islets with 15 and 30 mmol/liter glucose resulted in highly reproducible responses of
insulin release which were maintained for up to 5 hr.
Maximal response was elicited by theophylline, a standard test for measuring the functional integrity of the
islets (Lacy et al., 1972; Malaisse, 1973; Henquin and
Meissner, 1984).
Paraffin sections stained with immunohistochemical
techniques showed that the distribution of B, A, D, and
PP cells in the isolated islets is not different from that
of the intact islets; where A, D, and PP cells are located
at the periphery of the Fentral mass of B cells. Similar
immunohistochemical localization of B cells of the isolated islets was observed by Groth et al. (1980), Gray et
al. (19841, Warnock and Rajotte (19881, Delaby et al.
(1989), Kneteman et al. (1989), and Warnock et al.
(1989). There has been, to date, no detailed immunohistochemical study showing critical evaluation of
other endocrine non-B cells of the isolated islets. The
few isolated islets which were detected in the present
work containing a relatively large number of PP cells
were most probably derived from the lower part of the
494
M.M. EL-NAGGAR ET AL.
Fig. 4. An isolated pancreatic islet stained for insulin. Most of the
islet cells are positively stained. The peripheral cells show a strong
positive reaction, whereas the centrally located cells show a relatively
wcnk positivc rcnction. Immunopcroxidnsc stnin for insulin. X 660.
Fig. 6. A cells in an isolated pancreatic islet showing positive reaction (brown staining) to the anti-glucagon serum. These cells are located at the periphery of the islet. Immunoperoxidase stain for glucngon. X 660.
Fig. 5. Intact islet from normal rat pancreas showing positively
stained B cells in the central core of the islet surrounded by a mantle
of unstained non-B cells. Immunoperoxidase stain for insulin. X 650.
Fig. 7. Intact islet from normal rat pancreas stained for glucagon. A
cells show positive reaction (brown staining) and are arranged at the
periphery of the islet. Immunoperoxidase stain for glucagon. x 650.
juxtaduodenal pancreas, the latter was reported to be
rich in PP cells (see Baetens et al., 1979).
The results of the morphometric study indicated that
the cellular populations of the isolated islets are quantitatively different from those of the intact islets. The B
cells formed 92.74% of the volume and 90.75% of the
number of cells in the isolated islets. These were significantly higher than the percent volume and number
of the B cells of the intact islets, which were 66.04 and
70.32, respectively. On the contrary, the peripherally
arranged A, D, and PP cellular populations of the isolated islets showed significantly lower percent volumes
and numbers than those of the intact islets. Our quantitative data of the cellular populations of the intact
pancreatic islets are in agreement with those described
previously. According to Baetens et al. (19791, B, A, D,
and PP cells comprised 82.5, 15, 2, and 0.5% of the
volume of the intact dorsal pancreatic islets, whereas,
in the ventral pancreas, their percent volumes were 82,
1.3, 2.4, and 14.3, respectively. Hellman (1959), Carpenter and Lazarow (19671, and Dean (1973) have
shown that the centrally placed B cells comprise 6585% of the total volume of the intact pancreatic islets.
However, we could not locate in the available literature any quantitative study on the cellular populations
of the isolated islets.
The quantitative changes in the cellular populations
of the isolated islets were concomitantly accompanied
by a dramatic loss of the islet mass. Whereas the mean
true diameter of the isolated islets was 104.547 Fm,
that of the intact islets was 196.319 Fm. The loss of the
islet mass was uniform and affected all the size classes
of the isolated islets, as evidenced by shifting of their
size-frequency distribution towards the smaller diameters. The decrease in the percent volumes and numbers of A, D, and PP cells, which are located at the
periphery of the islets, indicate that the loss of the islet
mass during the isolation procedure had affected prin-
ISOLATED PANCREATIC ISLETS OF THE RAT
495
Fig. 8. An isolated islet stained for somatostatin. Positively stained
D cells are scattered a t the periphery of the islet. The section shows a
contaminating pancreatic acinus. Immunoperoxidase stain for somatostatin. x 650.
ery of the islet. This islet is most probably from the lower part of the
juxtadudenal pancreas (head) which is usually rich in PP cells. The
other islet, does not contain any PP cell. Immunoperoxidase for PP.
Fig. 9. An intact islet showing D cells with positive reaction (brown
staining) to the anti-somatostatin serum. The cells are scattered at
the peripheral part of the islet. Immunoperoxidase stain for somatostatin. x 650.
Fig. 11. Intact islet from the splenic part of normal rat pancreas.
PP-secreting cells appear positively stained with anti-PP serum. They
are few in number and scattered a t the peripheral part of the islet.
Immunoperoxidase stain for PP. x 650.
x 650.
Fig. 10. Two isolated islets stained for PP cells. One islet appears to
contain a good number of PP cells, which are scattered at the periph-
cipally the peripheral parts of the islets. This finding is
supported by the presence of irregular peripheral region which could be detected in most of the isolated
islets. Injury to the peripheral cells of the isolated islets
was also reported by Moskalewski (19651, Vance et al.
(1968), Buchanan and Mawhinney (1973), and Slavin
et al. (1977). Their observations were made, however,
without the support of morphometric quantitative
data. Our findings agree also with the observations of
Trimble et al. (1980), which described fewer A cells in
the intraportally transplanted islets as compared to the
islets within the normal pancreas. Vance et al. (19681,
and Buchanan and Mawhinney (1973) found that the
glucagon which is released from the isolated islets of
the rat was more variable than insulin. The variable
release of glucagon in the isolated islets was attributed
to damage of the A cells, which are situated more peripherally than the B cells. Slavin et al. (1977) attributed the injury of the peripheral parts of the islets to
the metabolic changes occurring in the islet cells during the isolation procedure. However, this injury could
also be due to enzymatic digestion of the peripheral
parts of the islets by the collagenase enzyme and mechanical trauma during the process of isolation.
Whether the changes in the normal percentage of the
endocrine cells of the peripheral parts of the isolated
islets can lead to changes in the hormonal secretory
function of the B cells, is not yet known. However,
there are various reports indicating the presence of
functional cooperation between various endocrine cells
496
M.M. EL-NAGGAR ET AL.
TABLE 3. The endocrine cell types of the isolated islets compared with those of the
intact islets'
Isolated islets
Volume density (percent volume)
B cells
A cells
D cells
PP cells
Percent No.
B cells
A cells
D cells
PP cells
Profile diameter of the islets (pm)
Intact islets
* 0.67 (240)
0.52 (331)
* 0.4 (205)
92.74
4.46 2
2.18
0.62 k
Remarks
*
66.04 0.39 (337)
19.38 k 0.71 (232)
6.09 & 0.21 (175)
8.49 0.49 (292)
P < 0.001
P < 0.001
P < 0.001
P < 0.001
70.32 2 0.25 (337)
18.77 t 0.71 (232)
4.23 0.13 (175)
6.68 t 0.58 (292)
133.15 1.37 (1036)
P < 0.001
P < 0.001
P < 0.001
P < 0.001
P < 0.001
*
0.18 (331)
*
*
*
90.75 0.95 (240)
5.82 0.68 (331)
2.45 t 0.42 (205)
0.98 0.23 (331)
75.41 t 1.53 (1107)
*
*
'Data are presented as means ? SEM.
Values between brackets represent the No. of the islets examined.
100
2
90
e
E
3
.Z volume of isolated islets
-
20
-
15
-
Z n u m b e r of isolated islets
% volume of i n t a c t islets
80
25
Z n u m b e r of i n t a c t islets
Isolated
islets
b0
Intact
v
70
L
60
ffl
4
#
I?
50
k
5
0
0
>
40
k
G
;
30
3
-
a)
e
E
3
20
5 -
z
0 -
e,
a
10
10
I I I I I I I I I
0
0
of the islet tissue, which plays an important role in
regulating the release of insulin in response to glucose.
Communication of the cells of the pancreatic islets occur through gap junctions. These junctions were reported to occur not only between B and B cells and
between A and A cells, but also between A, D, PP cells,
and adjacent B cells (Orci et al., 1973,1975; Unger and
Orci, 1981; Meda et al., 1983). Pipeleers et a1 (1982)
found that B cells in the isolated islets of the rat released 30 times more insulin in response to glucose as
compared with that released from purified single B
cells.
In conclusion, the results of the present work show
that the islets isolated by the enzymatic digestion technique have suffered considerable loss of their volume
during the isolation procedure. Most of the lost cells
were from the peripherally arranged A, D, and PP
cells, which led to quantitative changes of the cellular
populations of the islets. This might offer an explanation for the incomplete metabolic control which has
been a consistent finding in the short-term studies of
30
60
90
l l l l l , l j l l l , I
120
150
180 210 240
pancreatic islet transplantation, and the recurrence of
hyperglycemia in long-term studies (Steffes et al.,
1979; Trimble et al., 1980; Orloff et al., 1987). Evidently, to control diabetes mellitus by transplantation
techniques, isolated islet tissue with total cellular integrity seems to be essential. It, therefore, appears that
further refinements of the isolation techniques are necessary before a complete success could be achieved.
ACKNOWLEDGMENTS
We thank Prof. Ali Naji, Department of Surgery, Hospital of the University of Pennsylvania (Philadelphia,
PA), for his valuable help in developing the techniques
for this work. The authors gratefully acknowledge Mr.
Kabuye Sebastian and Mr. Mohamed Abdelrazak Saleeh for their excellent technical assistance and Mrs.
Kim Ishgi for her excellent secretarial help.
This work was supported by grant No. 171410 from
King Abdulaziz University, Jeddah, Kingdom of Saudi
Arabia.
ISOLATED PANCREATIC ISLETS OF THE RAT
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