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Department of Histology and Embryology, Biomedical Sciences Institute, University of São Paulo, São Paulo, Brazil 05508–900.
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • rats • weaning • gastric epithelium • cell proliferation • gastric growth
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Alvares and Gama 1993
), although different effects have
been reported, depending on the age studied (Alvares and Gama 1993
). In suckling pups, the proliferative process is highly
stimulated after food deprivation, whereas it is nonresponsive at
weaning and inhibited at 30 d (Alvares and Gama 1993
). During postnatal mo 1, the gastrointestinal mucosa of
rats undergoes structural and functional modifications that are related
to important dietary changes (Furihata et al. 1973
,
Henning 1981
, Ikezaki and Johnson 1983
,
Takeushi et al. 1981
).
There are few studies on the mechanisms that coordinate stomach growth.
However, they have been well explored in the small intestine; the
genetic program, diets and hormones interact in the control of
development (Lee and Lebenthal 1983
) by influencing
epithelial cell proliferation and differentiation (Boyle and Koldovsk
1980
, Lee and Lebenthal 1983
).
Because such processes are interdependent, Lin et al. (1998)
studied the effect of early weaning on DNA synthesis and
the activity of marker-enzymes in jejunum, showing that both are
stimulated by dietary manipulation. These changes are associated with
high plasma corticosterone levels (Lin et al. 1998
,
Yeh et al. 1986
), indicating that the dietary changes
interact with endocrine factors in the small intestine (Lee and Lebenthal 1983
, Yeh 1983
). In the stomach,
milk-borne hormones have also been suggested to influence cell
proliferation in suckling rats (Gama and Alvares 1996
).
However, it is not known whether all of the factors regulate the
proliferative responses of the gastric epithelium during postnatal
development.
The direct influences of starvation on cell proliferation and migration
of the gastrointestinal epithelia (Alvares and Gama 1993
, Gomes and Alvares 1998
) and evidence
suggesting that weaning is a period of intense changes (Alvares and Gama 1993
, Palanch and Alvares 1998
)
prompted us to inquire whether food deprivation effects are associated
with the type of diet ingested by the pups. Therefore, in this study,
we evaluated gastric cell proliferation by assessing both metaphasic
index and methapasic cell distribution in fed or food-deprived
rats, subjected to either early weaning or prolonged nursing
treatments. We also measured the thickness of the gastric mucosa and
recorded body weight, studying any possible growth alterations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wistar rats were housed in the Animal Headquarters (Instituto de
Ciências Biomédicas, São Paulo, Brazil) with natural
light from 
0600 to 1800 h. All cages with pregnant females were
checked frequently and birth was set as d 0. After 3 d, litters
were reduced to 8 pups (regardless of sex). Nonpurified diet (Rodent
Laboratory Chow, Purina Mills, Campinas, Brazil) and water were freely
available to dams and pups until the beginning of treatments. The
proximate nutrient composition of this diet is 30% protein, 6% fat
and 64% carbohydrate. This study was performed according to the
Procedures of the Animal Ethics Committee.
Dietary manipulation.
Early weaning. Two litters of 15-d-old pups were removed from their dams and placed in two short plastic cages (15 x 30 x 12 cm) with pine bedding. Because the pups were too young to nibble solid food, a container of powered nonpurified diet (mentioned above) and a small bottle of water were placed in the cage. Because pups might not defecate or urinate, these functions were stimulated by gently stroking their abdomens. Pups were killed after 3 or 7 d.
Prolonged nursing. Two litters were subjected to this condition at 15 d; four lactating females were used, two dams and two foster mothers. The dams and their litters were kept in cages without nonpurified diet to prevent the pups from reaching solid food. The foster dams were placed in other cages with free access to nonpurified diet. The females were exchanged every 12 h, to alternate feeding and nursing. In that way, each litter was nursed by its dam and a foster dam. The females were weighed daily and no loss was recorded after the alternative feeding schedule. Pups were killed after 3 or 7 d after.
Because there is no accurate way to quantify the food intake by pups subjected to early weaning or prolonged nursing, no attempt was made to measure this. However, body weights of the rats were recorded daily.
Food deprivation.
One day before killing, pups from a litter were randomly separated into
fed and 1-d food-deprived groups. Food-deprived rats were
placed in aluminum cages to avoid coprophagy for 20 h (at 17 d) or 24 h (at 21 d); such periods represent the time in
which neither milk nor food clots are observed in the stomach
(Alvares and Gama 1993
). The fed group was allowed to
suckle or feed until killing.
Therefore, there were four groups (n = 4) each of 18- and 22-d-old rats, as follows: early-weaned fed (EWF)3 ; early-weaned food-deprived (EWFD); prolonged-nursed fed (PNF); and prolonged-nursed food-deprived (PNFD).
Tissue preparation.
Eighteen- and 22-d-old pups were injected intraperitoneally with vincristine at a dose of 0.5 mg/kg body weight (Oncovin, Eli-Lilly, São Paulo, Brazil) at 0800 h and killed 2 h later. They were anesthetized with ether and the stomachs were collected and opened along the lesser curvature. Samples were taken from the corpus region, fixed in Bouin’s liquid and processed for histologic routine. Paraffin sections (4 µm) were stained with hematoxylin and eosin.
Metaphasic index (MI).
The MI, expressed as the number of metaphasic cells/total number of cells (%), was obtained by epithelial cell counting, which was performed by the same investigator who was unaware of the codes. At least 2500 epithelial cells were counted for each pup, in longitudinally sectioned glands examined by light microscopy (X800 field, Integrating Eyepiece I, Zeiss, Heidelberg, Germany).
The MI was obtained inside the proliferative compartment that comprises
the whole gland at 18 and 22 d as shown previously (Alvares and Gama 1993
).
Because early weaning and prolonged nursing might interfere with the
distribution of proliferative cells inside the compartment, we also
scored the metaphasic cells on a gland-positional basis, from the
top to the bottom. For that purpose, 100 metaphasic cells were counted
in randomly chosen glands (X800, Integrating Eyepiece II, Zeiss), which
were visually divided into regular intervals that allowed the correct
scoring of the positions of dividing cells (Ijiri and Potten 1983
).
Gastric mucosa thickness.
Gastric mucosa thickness was measured in four rats in each group by using an image analysis system (MINIMOP, Kontron, Heidelberg, Germany) and a Zeiss microscope (X400), to verify whether early weaning or prolonged nursing would lead to changes in growth. At least 15 fields were measured in each rat pup. The values were expressed in micrometers.
Statistical analyses.
Body weight, MI and gastric mucosa thickness are reported as means
± SD The MI were arcsin-transformed (Sokal and Rohlf 1995
) before statistical comparison. Two-way
ANOVA (diet x feeding state) was used to evaluate the data.
Differences between groups were analyzed by Tukey’s test with
P < 0.05 (GraphPad PRISM, Version 2.0, GraphPad
Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At 15 d, the body weight of the pups was considered normal (23.8
g) according to our laboratory standards. Table 1
shows body weights at 18 and 22 d, when rat pups were killed. A
cessation of weight gain was verified in the first 2 d of early
weaning (data not shown), so that at 18 d, pups that had been
weaned early were significantly lighter than the PNF group
(P < 0.05). After this initial period, pups that had
been weaned early recovered gradually at a rate of 2.5 g/d (data not
shown), and at 22 d, EWF rats were heavier than their PNF
counterparts (P < 0.05).
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Gastric mucosa thickness.
The gastric mucosa was well preserved after dietary manipulation and
injuries were not observed (Table 2
). There was no interaction between dietary manipulation and feeding
condition, suggesting that they independently affect the thickness of
gastric mucosa. When groups subjected to early weaned and prolonged
nursing were compared, significant differences were recorded among all
groups, except 18-d-old food deprived pups. Food deprivation affected
EWFD rats only at 18 d (P < 0.05).
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Metaphasic index.
Significant differences were found at 18 d (Fig. 1A
); there was an interaction between dietary manipulation and feeding
condition (P < 0.001), i.e., the effect of food
deprivation on gastric cell proliferation depended on the diet
condition. The MI of the EWFD group was significantly lower than that
of its EWF counterpart (P < 0.01), but such a
difference did not occur in rats subjected to prolonged nursing.
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) were
added to the test and compared with the current results (18-d-old rats:
1.77 ± 0.14 and 3.15 ± 0.53, for fed and food-deprived
groups, respectively). The MI of EWFD 18-d-old rats was significantly
lower than that of food-deprived pups (value above) (P
< 0.01). When a similar comparison was made among fed rats, the
MI of the EWF and PNF groups were greater than that of normally fed
pups (P < 0.01). At 22 d, the four groups did not
differ (Fig. 1B
). Frequency distribution of metaphasic cells.
Metaphasic cells were seen throughout the entire extension of the
gland, but some differences in their distribution were observed
(Fig. 2
). To compare such differences to the normal proliferative compartment
determined previously by Alvares and Gama (1993)
, their
curves are also shown in Figure 2A
, demonstrating that the
highest frequencies of metaphasic cells were recorded in the middle of
the gland. After early weaning (Fig. 2B
), however, the
curves were skewed to the left (middle to top of the gland), mainly in
EWF group. The prolonged nursing treatment (Fig. 2C
) kept
the highest frequencies in the middle of the gland, although the
pattern of the distribution curves was changed by increasing the values
at the shoulders. In the PNFD group, this effect was clearly seen,
whereas in the PNF group, only the tail was affected.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Early weaning and prolonged nursing of laboratory animals have been used since the early 1970s as models that provide important information on the role of diet in the development of the alimentary tract. In this study, we verified that some developmental variables were affected differently by food deprivation, depending on the dietary condition.
The body weight was affected by early weaning at 18 d and by
prolonged nursing at 22 d. These results are consistent with other
studies that reported body weight recovery 1 or 2 d after the
beginning of early weaning (Boyle and Koldovsk 1980
, Lerman and Koldovsk 1979
,
Niijar and Hatch 1991
). Niijar and Hatch (1991)
suggested that the abrupt change from milk to semiground
food resulted in starvation, which delayed body growth, as we also
observed when rats subject to early weaning and prolonged nursing were
compared. Boyle and Koldovsk (1980)
verified that
energy intake increases during d 2 postweaning, indicating the presence
of operative digestive processes that trigger weight gain. In our
study, pups at 18 d were recovering, and at 22 d they showed
a normal weight. For prolonged nursing, deleterious effects on growth
were also reported (Girard et al. 1992
, Grey et al. 1991
). Lebenthal et al. (1973)
suggested
that by the end of the milk feeding period, animals may have ingested
less energy than their littermates, and so begin to lose weight. Such
an observation may explain why PNF rats had lower weight than the EWF
group at 22 d. Food deprivation within the prolonged nursing
treatment only exacerbates weight loss, as we verified (Table 1)
. We
suggest that animals subject to prolonged nursing may be more
susceptible to the stress of food deprivation due to the lack of milk
(nutrient source).
Proliferative indices can be obtained by metaphasic index, as was done
in this study, or by other labeling methods such as tritiated thymidine
(autoradiography), or bromodeoxyuridine and other antigens
(immunohistochemistry). Alvares (1992)
showed that cell
proliferation could be evaluated equally well by the metaphasic or
labeling index. We used this technique recently (Gama and Alvares 1996
and 1998
) and the results suited cell
proliferation evaluation. Our current results showed that cell division
in the gastric epithelium is influenced differently by the diets at
18 d. The two-way ANOVA showed an interaction between dietary
manipulation and feeding condition, suggesting that food deprivation
may trigger different responses, depending on the nature of the diet.
We observed that when rats were food-deprived after a 3-d period of
early weaning, cell proliferation of the gastric epithelium was
inhibited, in contrast to the stimulus observed in 18-d-old pups under
normal dietary conditions (Alvares and Gama 1993
). This
inhibition exerted in pups that are weaned early is similar to that
found in food-deprived adult rats (Alvares and Gama 1993
, Hunt 1957
), indicating a change in the
response at 18 d promoted by the alteration in diet. Because this
result was obtained only in groups that are weaned early, it seems that
food deprivation stimulates cell proliferation only when 18-d-old rats
are fed normally, which at this age means milk and food. Curiously, at
22 d, the MI were not affected and no differences were detected
between food-deprived and fed pups. It is likely that at 22 d,
diet is no longer the major controlling factor for cell proliferation
in the gastric epithelium. We have already shown that 22-d-old rats
also do not respond to hormones (Gama and Alvares 1996
),
suggesting that other mechanisms may be operating at this transitional
stage.
The proliferative compartment comprises the whole extension of the
gland in rats of both ages studied and it is not affected by food
restriction (Alvares and Gama 1993
). However, we showed
here that metaphasic cells concentrate at the top of the gland after
early weaning, mainly in the EWF group. Such a peak is typical of adult
rats, suggesting that early weaning promoted the precocious appearance
of an adult feature.
Food deprivation effects did not depend on the nature of the diet
(Table 2)
. Our results indicate that such treatment promoted a
significant reduction only in rats that were weaned early at 18 d.
However, the comparison among groups suggested that depending on diet,
the thickness of the mucosa differs. Generally, after prolonged
nursing, we observed lower values. Such results are supported when the
current measurements are compared with those obtained previously for
normally fed pups (242 ± 10 and 240 ± 21 for fed and
food-deprived groups, respectively), and we observed that early
weaning thickened the gastric mucosa at 18 d, supporting body
weight and MI data. Thus, early weaning increases the thickness of the
gastric mucosa, whereas prolonged nursing decreases it. Similar results
were reported for the small intestine; Herbst and Sunshine (1969)
and Lin et al. (1998)
reported an
increase in the depth of the crypts after early weaning.
We demonstrated that when solid food was given at 15 d, food
restriction inhibited cell proliferation, a response that can impair
growth and lead to the precocious appearance of common adult features.
This can be explained by the role of diet (or its nature), i.e., at
18 d, the effects of food deprivation depended on what was being
ingested, milk, food or both. Different studies have shown that during
suckling, milk is not only a source of nutrients and antibodies, but
also a secretion rich in growth factors that influence the
proliferative and differentiation processes, in addition to protecting
the gastrointestinal tract (Carver and Barness 1996
,
Donavan and Odle 1994
, Olanrewaju et al. 1996
). Food deprivation is a physiologic stress, and also
represents the absence of an important modulatory agent of growth,
suggesting that after early weaning, not only is milk eliminated from
the diet, but also the control that it exerted. In fact, such
mechanisms are more complex because glucocorticoids seem to be
associated with precocious maturation as well (Lin et al. 1998
).
The prolonged nursing model is also useful in this kind of
investigation because it gives more information on the role of diet
during development. In this study, prolonged nursing did not have an
effect on the MI. Similar results were obtained by Grey et al. (1991)
and Lebenthal et al. (1973)
in cell
proliferation studies in the small intestine of rats. The distribution
of metaphasic cells along the gland was quite different when compared
with early weaning. At 18 d, the prolonged nursing effect was not
strong and the proliferative compartment remained in a transitory state
between the normal and early weaning patterns, whereas at 22 d,
the distribution was concentrated at the top of the gland. Thus,
prolonged nursing delayed the appearance of a well-defined
proliferative zone. This dietary condition also seems to retard growth,
as discussed earlier for the thickness of the gastric mucosa.
In conclusion, the present findings confirm our initial hypothesis that the effect of food deprivation on gastric epithelial cell proliferation depends on the nature of the diet. We suggest that during the suckling period, milk intake is a relevant modulatory factor for the proliferative processes in the stomach because such modulation can be disrupted in rats subjected to early weaning or prolonged nursing. During the 1st mo of postnatal development, this control is slowly modified, as indicated by the different effects exerted by dietary manipulation and food deprivation at 18 and 22 d. By the end of postnatal wk 3, factors other than diet may regulate cell proliferation in the gastric epithelium of rats.
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FOOTNOTES |
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3 Abbreviations used: EWF, early-weaned fed; EWFD, early-weaned food-deprived; MI, metaphasic index; PNF, prolonged-nursed fed; PNFD, prolonged-nursed food-deprived.
Manuscript received January 28, 2000. Initial review completed March 20, 2000. Revision accepted May 30, 2000.
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