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Variation in the silver staining of the Golgi complex along the epithelium of the intestinal villi in the adult rat.

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Variation in the Silver Staining of the Golgi Complex
Along the Epithelium of the Intestinal
Villi in the Adult R a t '
R. L. JOSEPHSON AND G. G . ALTMANN
Department of Anatomy, Health Sciences Centre, T h e University of
W e s t e r n Ontario, London 7 2 , Ontario, Canada
ABSTRACT
A light microscopic study was carried out on the Golgi complex
of the absorptive epithelial cells of the small intestine of adult male rats. Silver
impregnation methods (Da Fano's, Aoyama's, and Elftman's) were used. Deposits of silver in the Golgi complex were heaviest in the lower half of the villi.
Thereafter, a progressive diminution in the reaction occurred. In the uppermost
quarter of the villus no reaction was visible. Villi of the duodenum, midgut, and
terminal ileum all exhibited this phenomenon regardless of variations in fixation
method, concentration and pH of the silver solutions, exposure time to the silver
solutions, and various dietary regimens (high fat, no fat, and starvation). The
change observed in the silver reaction of the Golgi complex probably reflects a
structural or functional change of this organelle as the absorptive epithelial cells
migrate along the villi toward the extrusion zones.
The epithelium of the small intestine of (Da Fano, '20a,b); ( 2 ) Aoyama's silver
the rat is a rapidly renewing cell popula- method (Aoyama, '29); ( 3 ) Elftman's dition (Leblond and Stevens, '48). Its cells rect silver method (Elftman, '52). Silver
originate in the crypts of Lieberkuhn, mi- impregnation was carried out in the dark.
grate upward to the villus to form the ab- In all techniques, the tissues were emsorptive epithelium, and then move toward bedded in paraffin and sectioned longitudithe apex of the villus where they are ex- nally at 3-5 p .
Da Fano's method. Pieces of intestine
truded. During the migration of these cells
morphological and functional transfoma- were fixed in 15% formalin containing 1%
lions take place (Palade, '55; Padykula et cobalt nitrate for four to six hours at 24°C.
al., '61; Padykula, '62). As a part of a They were then transferred to a bath of
study of these transformations, the Golgi 1.5% silver nitrate for 24-36 hours at
complex was examined at the light micro- 24°C. Following a wash in distilled water,
scopic level using silver impregnation tech- the specimens were placed in Cajal's reniques.
ducing solution consisting of 2 % hydroquinone and 0.5% sodium sulphite in 15%
MATERIALS AND METHODS
formalin (Cajal, '12) for 20 hours at 24°C
Altogether 30 adult male Wistar rats ( 2 animals). A few variations were also
weighing 300-350 gm were used. Except tried: ( 1 ) the concentration of the silver
€or ten animals, all were maintained on solution was doubled to 3% silver nitrate
Purina Laboratory Chow. They were sac- while the exposure time of the tissues was
0 and
~ ~ ~P M
under held constant (1 animal); ( 2 ) the conrificed between 1
chloroform anaesthesia. Pieces of duode- centration was held at 1.5% while the
num (taken next to the pylorus), midpor- exposure time was doubled (1 animal);
tion of the small intestine, and terminal (3) both the concentration of the silver
ileum (taken next to the caecum), were solution and the exposure time of the tisremoved, cut open longitudinally, flattened sues to it were doubled (1 animal). Peron pieces of cardboard, and placed in the
Received Oct. 6, '71. Accepted Jan. 24, '72.
appropriate fixative for the following tech1 This work was supported by a grant of the Medical
niques: ( 1 ) the Da Fano silver method Research
Council of Canada to the second author.
ANAT. REC., 1'73: 221-224.
221
222
R. L. JOSEPHSON AND G. G . A L T M A "
fusion of fixative solution through the left
ventricle followed by immersion in fresh
fixative was also carried out ( 1 animal).
Aoyamds method. Pieces of intestine
were fixed in 15% formalin containing 1%
cadmium chloride for six hours at 24°C.
The use of cadmium chloride as a source
of divalent cations is the only essential
difference between this method and that
of Da Fano ( 2 animals).
Direct silver method. Specimens were
fixed and impregnated at the same time in
a solution of 15% formalin containing 2%
silver nitrate at 24°C. After two hours in
this solution, the samples were placed directly into a solution of 2% hydroquinone
in 15% formalin for two hours at 24°C.
They were then transferred into a 15%
formalin solution for 12 hours and processed for paraffin embedding the next
day (4 animals). In addition, a number
of variations were performed: (1) the
concentration of the silver solution was
doubled to 4% while exposure time was
held constant (1 animal); (2) the concentration was held constant while the exposure time was doubled (1 animal); ( 3 )
both the concentration and exposure time
were doubled (1 animal); ( 4 ) the pH of
the formalin-silver solution was varied between 4.0 and 7.2 in increments of 0.4 pH
(1 animal); (5) 2 cm segments of intestine were tied off, care being taken that
the blood supply to the segments remained
intact. The formalin-silver solution was
then injected into the lumen of the ligated
intestinal segments in situ (2 animals);
(6) tissue samples were washed for ten
minutes in TC Hank's solution (Difco
Laboratories, Detroit, Michigan) at room
temperature followed by immersion in the
formalin-silver solution ( 1 animal). Furthermore, a few experiments were carried
out in which the animals were placed on
special diets: ( a ) a diet rich in fat ( 3
animals); (b) a diet containing no f a t 3
( 3 animals); ( c ) starvation for 48 hours
prior to sacrifice (2 animals); and (d)
the same as in (c) but followed by gastric
intubation of 2 ml corn oil one hour prior
to sacrifice (as in Cardell et al., '67).
Only villi which were sectioned along
their longitudinal axis were considered.
The conditions for selecting villi cut along
their longitudinal axis were reported elsewhere ( Altmann and Leblond, '70).
RESULTS
All three silver methods revealed that
the silver reactivity of the Golgi complex
increased as the principal epithelial cells
ascended from the crypts of Lieberkuhn to
the villus. The epithelial cells on the lower
half of the villus showed maximal Golgi
impregnation (fig. 1, zone 1). Thereafter,
a progressive diminution in the reaction
occurred (fig. 1, zone 2). In about the
upper quarter of the villus, no Golgiassociated silver deposits could be seen
(fig. 1,zone 3). The extent of zone 3 varied
slightly from villus to villus. All three intestinal regions exhibited this phenomenon.
Aoyama's method appeared to be superior
to the Da Fano method in demonstrating
the reticular nature of the Golgi complex.
However, the direct silver method in the
opinion of the authors is by far the best
method in both its reliability and its simplicity. Perfusion and intraluminal injection of fixative resulted in better preservation of tissue architecture, although there
was no observable difference in the impregnation characteristics of the Golgi
complex from those seen with conventional
fixation procedures. In all cases, increasing
the concentration of the silver solution or
the duration of tissue exposure to it, did
not change the appearance of the zones
shown in figure 1. Varying the pH of the
formalin-silver solution in the direct method also resulted in no observable differences. Washing tissue samples in Hank's
solution prior to fixation, largely eliminated a layer of debris over the tip area
which was otherwise impregnated with
silver. Again, no changes in the Golgiassociated silver deposits were observed.
The various dietary regimens did not alter
the Golgi impregnation characteristics of
the absorptive cells. It seemed, however,
that the diet had a part in causing an infranuclear region of reduced silver in absorptive cells in the upper one third of the
villus (fig. 2). All animals except the ones
2 Ground Purina Laboratory Chow enriched with
corn oil (40% by weight).
3 The comaosition of the diet (o/, bv weieht) was
as follows: 34% casein; 8% lactalbimin; -30% s<
crose; 20% corn starch; .8% ,alphacel; 2% salt mixture 2 U.S.P.:2% vitamin diet fortification mixture.
All the materials were obtained from Nutritional Biochemicals Co., Cleveland, Ohio.
223
SILVER STAINING OF THE GOLGI COMPLEX
Fig. 1 A whole villus is shown. Composite
picture. Zone I shows maximal Golgi reaction. In
zone 11, the reaction progressively diminishes. No
reaction is visible in zone 111. The extrusion zone
( E l is well delineated by about six darkly stained
cells at the villus tip. The change in the silver
staining of the Golgi complex is apparent much
below the extrusion zone. Da Fano’s method.
x 225.
Fig. 2 Upper two-thirds of a villus is shown.
In the upper third of the absorptive epithelium,
the hfranuclear accumulation of silver ( D ) is
visible. zones11 and 111 are also visible. Goblet
cg) and argentaffin
( a ) cells exhibit dense
deposits’
method’
450’
224
R. L. JOSEPHSON AND G. G. ALTMANN
which received no food 48 hours prior to be changes which indicate a structural or
sacrifice, exhibited this infranuclear ac- functional transformation of the organelle
cumulation of silver.
as the absorptive epithelial cells progress
along the villi toward the Extrusion zones.
DISCUSSION
A progressive differentiation of the normal intestinal epithelium occurs during
the migration of the epithelial cells from
the crypts of Lieberkuhn to the extrusion
zone (Padykula, '62). The changes observed in the silver-reactivity of the Golgi
complex may be a morphological expression of these changes. The possibility of
the zonation being of artifactual origin is
unlikely in view of the fact that the results
were not altered by variations in fixation
technique or by washing the samples in
Hank's solution.
Change in the Golgi complex of the intestinal epithelial cells has been observed by
a number of authors. For example, it has
been shown that the Golgi apparatus becomes more conspicuous as the epithelial
cells migrate to the villi (Palade, '55; Padykula, '62). Furthermore, diminution of
Golgi-associated substances has been demonstrated by EM radioautography utilizing
galactose-3Has a label for glycoprotein synthesis in rat intestinal epithelial cells. All
villus columnar cells were seen to incorporate label in the supranuclear Golgi region but the intensity of the reaction decreased gradually from the base to the apex
of the villus (Bennett, TO).
Since the mechanism of silver impregnation is not well understood, it is not possible at present to state the exact sites or
materials which are stained by silver. It is
believed by some authors that phospholipids and possibly bound proteins are
stained (Elftman, '53; Thompson and
Hunt, '66; Pearse, '68). It is therefore possible that the silver deposits were associated with the membranes of the Golgi
complex. The exact nature of this reaction,
however, is to be clarified by further
studies.
In conclusion, it appears that changes
occur in the Golgi complex as the principal
cells migrate along the villus. These
changes are reflected in the silver reaction. They do not seem to be dependent
on dietary conditions and may therefore
LITERATURE CITED
Altmann, G. G., and C. P. Leblond 1970 Factors influencing villus size in the small intestine of adult rats as revealed by transposition of intestinal segments. Am. J. Anat., 127:
15-36.
Aoyama, F. 1929 Eine Modification der Cajalschen Methode zur Darstellung des Golgischen
binnennetz Apparate. Ztschr. Wissen. Mikr.,
46: 489-491.
Bennett, G. J. 1970 Migration of glycoprotein
from Golgi apparatus to cell coat in the columnar cells of the duodenal epithelium. J. Cell
Biol., 45: 668-673.
Cajal, S. R. Y. 1912 Formula de fijacion para
la demonstracion facil del apparato reticolar de
Golgi y apuntes sobre la disFosicion de dicho
apparata en la retina en 10s nervios y algunos
estados patologicos. Trab. del lab. de invest.
biol. de la Univ. de Madrid, 10: 209-220.
Cardell, R. R., S . Badenhausen and K. R. Porter
1967 Intestinal triglyceride absorption i n the
rat. A n electron microscopical study. J. Cell
Biol., 34: 123-155.
Da Fano, C. 1920a Method for the demonstration of Golgi's internal apparatus. J. Physiol.,
53: xcii-xciv.
-1920b Method for the demonstration
of Golgi's internal apparatus in nervous and
other tissues. J. Roy Micr. SOC.,40: 157-161.
Elftman, H. 1952 A direct silver method for
the Golzi apparatus. Stain Techn., 27: 47-52.
1953 Response of the Golgi material to
chemical reagents. J. Histochem. and Cytochem., I: 387-388.
Leblond, C. P., and C. E. Stevens 1948 The
constant renewal of the intestinal epithelium in
the albino rat. Anat. Rec., 100: 357-378.
Padykula, H. A. 1962 Recent functional interpretations of intestinal morphology. Fed. Proc.,
21: 873-879.
Padykula, H. A., E. Strauss, A. J. Ladman and
F. H. Gardner 1961 A morphological and
histochemical analysis of human jejunal epithelium in nontropical sprue. Gastroent., 40:
735-765.
Palade, G . E. 1955 A small particulate component of the cytoplasm. J. Biophys. Biochem.
Cytol., I: 59-68.
- 1958 A small particulate component of
the cytoplasm. In: Frontiers in Cytology. S. L.
Palay ed. Yale University Press, New Haven,
Conn., pp. 283-304.
Pearse, A. G. 1968 Histocheniistry. Theoretical
and Applied. Third ed. Vol. 1. J. and A.
Churchill Ltd., London W1, pp. 78-79.
Thompson, S. W., and R. D. Hunt 1966 Selected
Histochemical and Histopathological Methods.
Charles C Thomas, Springfield, Ill., pp. 805-809.
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adults, alone, complex, epithelium, silver, intestinal, variation, ville, golgi, rat, staining
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