Egypt. J. Histol. Vol. 33, No. 3, Sep., 2010: 479 - 488
(ISSN: 1110 - 0559)
Original Article
Effect of the Overcrowding on Fundus of Stomach in Adult Male Albino
Rats with Special Reference to the Protective Role of Bran
Hany K. K. Mostafa
Histology Department, Faculty of Medicine, Ain Shams University
ABSTRACT
Introduction: Overcrowding as a chronic source of stress constituted a major threat to psychological well-being. Dense
populations were characterized by considerably increased aggressive behavior.
Aim of the Work: Is to shed the spot on the effect of overcrowding stress on fundic glands and the way to antagonize
by the use of wheat bran.
Materials and Methods: Fifteen animals were used in this study divided into two groups: control group (group I)
was formed of 5 animals and group II was formed of 10 animals that were subjected to overcrowding. Group II was
further subdivided into two subgroups IIa: 5 animals were subjected to overcrowding, subgroup IIb was formed of
5 animals that were given bran orally together with the stress procedure. The duration of experiment was 7 days. at
the end of the experiment stomach specimens were obtained, processed and subjected to different histological and
immunohistochemical techniques.
Results: Overcrowding stress led to desquamation, necrosis and ulceration of gastric epithelium. Mononuclear cellular
infiltrate and congested blood vessels were also noted in lamina propria. Parietal cells were decreased in number while
mucous neck cells were increased in fundic glands. Acid mucous produced by mucous neck cells was increased more
than neutral mucous produced by surface mucous epithelial cells. E cadherins were decreased in intensity in surface
mucous epithelial cells led to disturbed barrier against gastric acidity. Subgroup IIb showed picture similar to that of the
control. The previous data were confirmed by morphometric and statistical analysis.
Negative Glial-Fibrillary-Acidic Protein (GFAP) reaction in the full term filum terminale group. In two weeks group;
there was positive reaction. In one month group there was strong reaction which persisted with gradual decline in three
and six month groups. In one year, GFAP reaction was scanty positive or even negative.
Conclusion: It could be concluded that bran has great protective value against gastric mucosal damage caused by
overcrowding stress. It is recommended that high fiber diet in meals especially in cities with dense population is needed
to decrease gastric mucosal damage.
Key Words: Overcrowding, bran, fundic glands, E
Corresponding Author: Hany K.K. Mostafa
cadherins.
Tel.: 0119717122 E-mail: [email protected]
INTRODUCTION
Crowding as a chronic source of stress constituted
a major threat to psychological well-being. Crowding
results in anxiety and social instability. Dense populations
were characterized by considerably increased aggressive
behavior. Overcrowding was considered one of the most
important problems in many cities in different countries
of the world. It disturbed the homeostasis and might
induce various disorders1. Crowding became one of the
most popular stressors in experimental medicine, since it
could be regulated easily and was reproducible2.
hypertension, arteriosclerosis, diabetes mellitus and
other diseases in clinical and epidemiological studies4.
Preventative medicine is targeting chronic diseases
such as heart disease, cancer, diabetes and obesity. Intake
of dietary fiber has beneficial effects on the risk factors
for developing several chronic diseases. Populations
that consume more dietary fiber became less vulnerable
to many chronic diseases. Dietary reference intakes
recommended the consumption of 14 g of dietary fiber
per 1000 kcal, or 25 g for women and 38 g for men,
based on the goal of protection against cardiovascular
disease5. The fiber diet chosen for this study, wheat bran,
is important fiber source that has different compositions
and physicochemical properties. Wheat bran is fermented
slowly, resulting in greater concentrations of butyrate
Dense populations could influence the hypothalamicpituitary-adrenocortical axis, the hypothalamic-pituitarygonadal axis, sympatho-adrenomedullary system and
sympathetic nervous system, leading to changes in many
organs3. Overcrowding stress had been implicated in
42 (1207-2010)
479
Hany K. K. Mostafa
in the gastrointestinal tract6. However, because of the
decreased fermentability of wheat bran, it maintains a
greater dilution potential and fecal bulking capacity.
Dilution potential was believed to be important in
preventing many gastrointestinal tract cancers by
reducing the exposure of the surface epithelium to
carcinogens and promoters7.
Subgroup IIa: Was formed of 5 rats exposed to
overcrowding stress.
Subgroup IIb: Bran intake before and during
crowding stress
This subgroup included the animals which were
subjected to crowding condition (stress for 7 days). They
were housed in the previously described small cages for
4 hours / day for 7 days. This subgroup was given bran
in their diet during the stress procedure. The wheat bran
was supplied at a dose of 10 gm / 100gm of ad libitum
diet after it was dissolved in suitable amount of distilled
water10. Wheat bean was supplied as a powder from
Cairo mills.
The apical part of surface mucous epithelial cell
forms tight junctions with each other. Tight junctions are
formed of many integral proteins. Cadherins is one of the
most important proteins in tight junctions in epithelium.
It is calcium dependant protein reinforce the junctions
between two adjacent cells. So it plays important role in
the barrier of stomach8.
AIM OF THE WORK
After the last four hours of crowding stress, the
animals were sacrificed at the appropriate time by
decapitation under thiopental sodium phosphate
(obtained from E.P.I.CO.) in a dose of 40mg/kg body
weight intraperitoneally calculated according to Paget
and Barnes equation11. The abdomen was opened and
stomach was excised. It was opened along its greater
curvature, washed gently with saline. Each specimen
was fixed in 10% formol saline, dehydrated, cleared
and paraffin sections were prepared as usual and were
subjected to the following stains:
The aim of this work to shed the spot on the effect
of overcrowding stress on fundic glands and the way to
antagonize by the use of wheat bran.
MATERIALS AND METHODS
Fifteen adult male albino rats with an average weight
of 200 gm were used in this study. All rats were first put
in control stainless steel cages with galvanized iron wire
gauges. The size of the control cage was 41X28X19 cm
which allowed the animals to move freely. All animals
were supplied with drinking water and ample amounts
of food. They were handled daily for one week prior to
the experiment to minimize the non specific stress on the
days of the experiment.
1. Histological study12:
A. Haematoxylin and Eosin.
B. Alcian blue-PAS stain.
2. Immunohistological study13:
Avidin biotin Immunohistochemical staining for E
cadherin.
Stress Protocol9:
The stress procedure involved placing group of five
male rats in cage of size 31x18x10 cm in such a way
that only minimum mobility was possible inside the
cages. The rats were kept in this overcrowded condition
continuously for 4 hours / day then they were shifted
to the control cages. All experimental stress procedures
started between 9:00 AM and end at 1:00 PM to minimize
the effect of circadian rhythm of hormones.
Avidin-biotin
peroxidase
staining technique:
immunohistochemical
Formalin fixed paraffin embedded sections were
employed for immunoperoxidase staining of the stomach
using avidin-biotin peroxidase complex method.
This technique was used to detect E cadherin in
zonula adherentes and occludentes of surface mucous
epithelial cells of the stomach. Primary antibodies
were used against E cadherin. Sections were put on
positively charged glass slides. The paraffin sections
were deparaffinized, hydrated and then put in 10% H2O2
to block endogenous peroxidase activity. Unmasking of
antigenic sites was carried out by transmitting sections
into a jar containing 0.001M citrate buffer (PH 6). Then
boiling in microwave for 4 minutes at temperature grade
VIII followed by 2 minutes at grade II. Incubation with
1/100 normal rabbit serum for 20 minutes was done in
order to block non-specific background staining. The
diluted primary antibody was then added on the slides
and kept for 2 hours. The slides were then washed with
Animal Groups:
Group I: The Control Group:It is consisted of 5 rats which were kept under standard
laboratory conditions without any stress exposure.
Group II: Crowding Stress Group:Included 10 animals which were subjected to acute
crowding condition (stress for 7 days). They were housed
in the small cages (31x18x10) cm for 4 hours / day for 7
days according to stress protocol mentioned before. This
group was further subdivided into two subgroups 5 rats
each:
480
Effect of the Overcrowding on Fundus of Stomach in Adult Male Albino Rats with Special Reference to the Protective Role of Bran
phosphate buffer and 1/400 biotinylated rabbit anti-goat
IgG secondary antibody was then added to the slides and
kept for one hour. Streptavidin peroxidase was then added
to bind with biotin residue on the linking antibody for 10
minutes. Washing was then done by phosphate buffer.
The site of antibody immunostaining was visualized
by avidin-biotin peroxidase complex (ABC reagent)
for 30 minutes. Freshly prepared diaminobenzidine
(DAB) was used as chromogen. Sections were incubated
with DAB for 10 minutes then washed with tap water,
counterstained with haematoxylin, dehydrated and
mounted. For negative control, the primary antibody was
replaced by phosphate buffered saline.
gland characterized by their highly acidophilic cytoplasm
and central rounded nuclei (Fig. 4). Chief cells that lined
the base showed basal basophilia, apical acidophilic
cytoplasm and basal nuclei. The lamina propria of the
stomach was composed of loose connective tissues
interspersed between gastric glands together with
connective tissue cells and smooth muscle fibers that
separated the mucosa from the underlying submucosa.
The surface of the mucosa appeared to be covered by
a thin film of mucus coat which was composed of PAS
stained areas and Alcian blue stained material. These
materials extended to fill the pits and lumina of isthmus
of glands (Fig. 5).
Results: Cells contained E cadherins within their cell
membrane appear brown and nuclei blue.
B) Immunohistochemical Results
Immunohistochemical stain was used for
demonstration of E cadherin at the lateral border of
surface mucous epithelial cell. E cadherin were found
in the zona occludentes and zona adherentes. The cell
membrane of epithelial cell showed dense brown stain
(Fig. 6). The reaction appeared as dark brown granules.
3. Morphometric Study:
The following items were measured in 5 non
overlapping fields in 5 different sections of the stomach
in each animal in the different subgroups.
1. The mean number of parietal cells in fundic glands
of stomach in H and E stained sections.
2. Mean thickness of Alcian blue areas in Alcian bluePAS stained sections (Fig. 1).
3. Mean thickness of immunostaining positive for E
cadherin stained sections (Fig. 2).
Group II
Subgroup IIa: Overcrowded Subgroup
A) Histological Results
The fundic mucosa of animals of this subgroup
showed areas of discontinuity of surface mucosa and
other areas of erosion at pits of the mucosa. There was
distortion of normal architecture of fundic glands with
invasion by mononuclear cells (Fig. 7). The blood vessels
in different layers of fundus were congested as well as
mononuclear cellular infiltration could be seen within the
lamina propria (Fig. 8). Moreover, the mucosa showed
ulceration of the surface and vacuolation of some parietal
cells (Fig. 9).
The measurements were done by using image
analyzer (Leica Q 500 MC program) in the Histology
Department, Ain Shams University.
3. Statistical Study:
Student’s “t” test was used to compare the data and the
P value was calculated using SPSS program. Statistical
significance was determined at a level of P < 0.05. So,
P>0.05 was considered non-significant and P value <0.05
was considered significant. (n = 5).
In PAS-Alcian blue stained sections, the mucosa
showed apparent increase in Alcian blue stained mucous
more than PAS stained mucous in comparison to that of
the control (Fig. 10).
RESULTS
Control Group:
A) Histological Results
B) Immunohistochemical Results
Examinations of sections of the fundus of stomach
of control rats; showed that the wall of the fundus of the
stomach was formed of 4 classic layers namely, mucosa,
submucosa, musculosa and serosa. Gastric mucosa was
consisted of surface epithelium that extended into the lamina
propria forming pits or foveolae, at the bottom of which
open one or more glands. Each gland was divided into three
regions: isthmus, neck and base (Fig. 3). The fundic glands
were simple branched tubular with different types of cells
lined the pit and glandular regions. Surface mucous cells
lined the gastric pits; they were columnar acidophilic cells
with oval basal nuclei (Fig. 4). The neck of the glands was
lined by mucous neck cells. They appeared as groups of
cuboidal cells with flattened basal nuclei and vacuolated
cytoplasm. Parietal cells are dispersed throughout the
Immunostaining for E cadherin showed faintly
stained areas at the lateral borders of surface columnar
epithelial cells in gastric mucosa. Zones of E cadherin
were narrow and faint on surface mucosa (Fig. 11).
Subgroup IIb:
A) Histological Results
Animals of this subgroup were subjected to
overcrowding. Bran was added to the food of the animals.
Examination of sections of the gastric mucosa of
animals of this subgroup revealed that it was almost
similar to that of the control with no surface erosions
or ulceration. Surface mucous epithelial cells appeared
481
Hany K. K. Mostafa
normal with no areas of epithelial discontinuity and
extended to line the pits and isthmus. Parietal cells
appeared normal with acidophilic cytoplasm and widely
distributed among the other cells (Fig. 12). The apical
part of the gland was crowded by PAS positive mucous
granules. The mucous neck cells at the neck of the fundic
glands are moderately stained by cytoplasm by Alcian
blue stain (Fig. 13).
B) Immunohistochemical Results
The cell membrane of mucous epithelial cell showed
apparent dense brown stain (Fig. 14). The intensity of the
E cadherin was high as that in the control group.
Morphometric and statistical analysis:
Table 1: Showed the mean number of parietal cells in fundic
glands (High power X40) stained by H & E in different
subgroups:
Group I
Subgroup IIa
Subgroup IIb
Mean
29.6
22.4
27.2
SD
2.302
1.817
1.924
T test
5.49
1.78
P value
0.001
0.111
S
NS
Significance
Histogram 2: Showed the mean thickness (in µm) of mucous neck cell
zone in fundic glands (medium power X20) stained by Alcian blue-PAS
different subgroups:
Table 3: Showed the mean thickness (in µm) of E cadherin
in surface mucous epithelial cell (medium power X 20)
stained by immunostaining in different subgroups:
Group I
Subgroup IIa
Subgroup IIb
Mean
71.4
42.2
72.8
SD
6.877
5.675
4.438
T test
7.32
0.38
P value
0.000
0.712
S
NS
Significance
Histogram 1: Showed the mean number of parietal cells in fundic
glands (high power X40) stained by H & E in different subgroups:
Table 2: Showed the mean thickness (in µm) of mucous neck
cell zone in fundic glands (medium power X20) stained by
Alcian blue-PAS in different subgroups:
Group I
Subgroup IIa
Subgroup IIb
Mean
56.6
111.6
59.2
SD
2.881
5.941
1.483
T test
18.62
1.79
P value
0.000
0.111
S
NS
Significance
Histogram 3: Showed the mean thickness of E cadherin in surface
mucous epithelial cell (medium power X20) stained by immunostaining
in different subgroups:
482
Effect of the Overcrowding on Fundus of Stomach in Adult Male Albino Rats with Special Reference to the Protective Role of Bran
Fig. 1: Showing demonstration for measurement of thickness of alcian
blue stained (AB) areas in PAS – Alcian blue stained sections.
Fig. 4: Showing surface columnar epithelial cells (SME) extending to
line the gastric pits and isthmus. The glands are lined by different types of
cells: mucous neck cell (MN) and parietal cell (PC). Notice the pits and
parts of fundic glands: isthmus, neck and base.
Control group
H&E X 640
Fig. 2: Showing demonstration for measurement of thickness of
immunostaining for E cadherin in Avidin Biotin peroxidase for E cadherin
stained sections.
Fig. 5: Showing surface columnar cells with intensely PAS stained
mucous (PAS). While few areas are occupied by Alcian blue stained
mucus granules (AB).
Control group
PAS - Alcian blue stain X640
Fig. 3: Showing the mucosa of the stomach with pits and glands. The
fundic glands are formed of isthmus, neck and base. Notice the underlying
muscularis mucosa (mm).
Control group
H&E X 250
Fig. 6: Showing intensely stained E cadherin mainly at the lateral borders
of surface columnar epithelial cells in gastric mucosa with thick and
dense areas of brown granules at the cell membrane (↑).
Control Group
Avidin Biotin peroxidase for E cadherin. Counterstained with H X640
483
Hany K. K. Mostafa
Fig. 7: Showing distortion of the normal architecture of fundic glands (↑).
The lamina propria near the base shows mononuclear cellular infiltration
(MN).
Subgroup IIa
H&E X 640
Fig. 9: Showing ulceration of the surface mucosa (U) and vacuolation of
some parietal cells (↑).
Subgroup IIa
H&E X 640
Fig. 8: Showing mononuclear cellular infiltration (MN) at lamina propria.
Congested blood vessels (cong) are seen in different layers of the stomach.
Subgroup IIa
H&E X 640
Fig. 10: Showing increased Alcian blue stained mucous (AB) with
decreased PAS stained mucous (PAS).
Subgroup IIa
PAS - Alcian blue stain X640
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Effect of the Overcrowding on Fundus of Stomach in Adult Male Albino Rats with Special Reference to the Protective Role of Bran
Fig. 11: Showing faintly stained E cadherin (↑) at the lateral borders of
surface columnar epithelial cells in gastric mucosa.
Subgroup IIa
Avidin Biotin peroxidase for E cadherin. Counterstained with H X640
Fig. 12: Showing the structure of fundic mucosa similar to that of the
control.
Subgroup IIb
H&E X 640
485
Fig. 13: Showing surface columnar cells with intensely stained
PAS +ve material (PAS). While few areas are faintly stained by Alcian
blue (AB). They are nearly similar to that of the control.
Subgroup IIb
PAS - Alcian blue stain X640
Fig. 14: Showing intensely stained E cadherin (↑) mainly at the lateral
borders of surface columnar epithelial cells in gastric mucosa. It shows
intense areas of brown granules.
Subgroup IIb
Avidin Biotin peroxidase for E cadherin. Counterstained with H X640
Hany K. K. Mostafa
DISCUSSION
overcrowding. It was recorded that crowding induced
oxidative damage of surface epithelium18,19. This
postulation was confirmed by others who stated that
crowding induced generation of reactive oxygen radicals
in the fundic glands of the affected rats20. Another
etiological factor was also supposed by biochemical
investigators who recorded in their study that a decline
in activities of many endogenous antioxidant enzymes
exacerbating oxidant mediated tissue injury21.
Overcrowded areas were one of biggest problems in
the world. The authorities in different countries put plans
and budgets for treating or even shrink this problem.
In this experiment the overcrowding stress resulted
in marked affection of fundic glands of adult male albino
rats. The findings of the present work were in consistence
with the clinical findings of many investigators who
reported in their study that prevalence of gastric
symptoms like heart burn, epigastric pains, nausea and
vomiting were increased in areas of dense populations14.
In subgroup IIb rats exposed to overcrowding stress
but with bran intake, there were histological picture and
immunohistochemical picture almost similar to that
of the control. The mucosa of fundus showed normal
fundic glands no ulceration or desquamations. Mucous
content and barrier junctions became similar to that
of the control. These findings were in accordance to
previous authors22 who explained that bran have a
healing effect on epithelium of the stomach by increased
barrier thickness and restored cyclooxygenase enzyme
activities. Prostaglandins balance stimulated epithelium
to produce normal mucous together with bran fibers
that protect the mucosa from any insult23. Further more
other workers supposed that bran fibers contacts with
surface mucosa helped production of regulatory factors
that helped rapid regeneration of mucosa24. In addition,
bran mainly augments the normal defensive mechanisms
and homeostasis of the stomach25. Bran also restored the
antioxidant enzymes activities so it hasten the clearing
of reactive oxygen radicals consequently abolishes any
hazardous effects produced by overcrowding stress. This
agreed with some authors26 who postulated bran as byproduct of wheat milling but it contains antioxidants,
vitamins, minerals, nutritive in addition to the usual
fibers content.
These findings were revealed as ulcerations, necrosis,
desquamation and distorted fundic glands. These were
in agreement with some researchers15 who explained
that crowding stress led to increased glucocorticoids as
it disturbed hypothalamo-hypophyseal-pituitary axis.
Glucocorticoids led to inhibition of cyclooxygenase
enzymes that proved to be responsible for prostaglandins
production. Several components of mucosal defense
were influenced or mediated by prostaglandins. Other
workers said that the mucosa of crowded subgroup
showed marked reduction of oxidative phosphorylation
enzymes so cell metabolism became severely affected17.
Crowded subgroup showed increased acid mucous
relative to neutral mucous. Significant increase in mucous
neck area and decrease in number of parietal cells were
observed in comparison to the control group. These
findings agreed with some authors16 who reported that
overcrowding could lead to many defensive mechanisms
as increased number of mucous neck cells and decreased
number of parietal cells. These are defensive mechanisms
to overcome the state of damage generated by crowding
stress.
CONCLUSION
This from this study it could be reported that bran has
great protective value against gastric mucosal damage
caused by overcrowding stress. It is recommended that
high fiber diet in meals especially in cities with dense
population is essential in order to decrease gastric
mucosal damage.
Moreover congested blood vessels and mononuclear
cellular infiltration were also observed in crowded
subgroup. This went hand by hand with some authors17
who postulated that crowding affected the mucous
barrier and acidity of the stomach with subsequent
bacterial invasion. These bacteria were chemotactic for
mononuclear cells and also caused congestion of blood
vessels.
Further research: Complete analysis for different
components of wheat bran is recommended to know the
exact active ingredient that may have role in improvement
of digestive system performance.
Crowded subgroup also showed decrease in E
cadherin of tight junctions between epithelial mucous
cells. Significant decrease in thickness of areas of E
cadherin of crowded subgroup in comparison to that
of the control was observed. These findings were in
accordance with other workers18 who said that crowded
environment could alter the intercellular junctions due
to alteration in PH of the gastric lumen and disturbed
metabolism of surface epithelial cells.
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487
‫‪Hany K. K. Mostafa‬‬
‫الملخص العربى‬
‫تأثير الضغط الناتج عن الزحام الشديد على قاع معدة ذكور الجرذان البيضاء البالغة مع اإلشارة للدور الوقائى للردة‬
‫هانى كمال كامل مصطفى‬
‫قسم الهستولوجيا ‪ -‬كلية الطب ‪ -‬جامعة عين شمس‬
‫المقدمة‪ :‬يعتبر الزحام الشديد من الضغوط الجسيمة المزمنة فى الحياة اليومية كما أنه يعد تهديدا أساسياً للصحة النفسية‪ .‬وقد بدا‬
‫واضحاً أن المجتمعات شديدة الكثافة السكانية هى المجتمعات األكثر عدوانية‪ .‬مما تقدم‪ ،‬كان الغرض من هذا البحث هو إلقاء‬
‫الضوء على تأثير الزحام الشديد على قاع المعدة مع المحاولة للتغلب على هذه التأثيرات من خالل استعمال الردة‪.‬‬
‫المواد والطرق‪ :‬استعمل فى هذه الدراسة ‪ 15‬جرذاً ابيض مقسمة كالتالى‪ :‬المجموعة األولى مكونة من ‪ 5‬جرذان وهى‬
‫المجموعة الضابطة‪ ،‬المجموعة الثانية هى المجموعة التى تعرضت لضغط الزحام الشديد وهى مكونة من ‪ 10‬جرذاً ابيضاً‪ .‬كما‬
‫تم تقسيم المجموعة الثانية ملحقاً إلى ‪ 2‬مجموعة فرعية ‪ 5‬جرذان بكل مجموعة‪ :‬الثانية الفرعية أ وهى تعرضت لضغط الزحام‬
‫الشديد فقط‪ ،‬المجموعة الثانية الفرعية ب وهى تعرضت لضغط الزحام الشديد مع أضافة الردة فى غذاء هذه المجموعة الفرعية‬
‫وقد كان زمن هذه التجربة ‪ 7‬أيام‪ .‬وبنهاية زمن الدراسة تم جمع الحيوانات و تخديرها عند وقت التضحية وتم إستخراج المعدة‬
‫و تجهيزها ومعالجتها بتقنيات هستولوجية وهستوكيميائية مناعية‪.‬‬
‫النتائج‪ :‬بفحص مقاطع المعدة المأخوذة من المجموعة الثانية أ مجموعة الزحام الشديد تبين أن هذا الضغط قد أدى إلى تنكرز‬
‫وتقشرالنسيج الظهارى مما يؤدى إلى تكون قرح فى الطبقة المخاطية لجوف المعدة‪ .‬أما الصفيحة األساسية قد أظهرت احتقان‬
‫فى االوعية الدموية وتخلل للخاليا وحيدة النواة‪ .‬هذا باإلضافة إلى قلة عددالخاليا الجدارية وزيادة سٌ مك الجٌ زء المٌغطى بالخاليا‬
‫المخاطية العنقية وكان ذلك ذا داللة إحصائية‪ .‬وقد أدى ذلك لزيادة صبغة األلشيان االزرق بالنسبة لصبغة حامض البريوديك‬
‫صبغت بالصبغتين‪ .‬أما بالنسبة للمقاطع المصبوغة بتقنيات هستوكيميائية مناعية فقد قلَت كثافة بروتين‬
‫والشيف فى المقاطع التى ُ‬
‫اإل‪ -‬كادهرينز فى خاليا السطح المخاطية العمودية مما يضعف قوة حاجز المعدة فى هذه المجموعة‪ .‬أما المجموعة الفرعية‬
‫الثانية ب فقد أظهرت نتائج مشابهة للمجموعة الضابطة‪ .‬وقد تم تأكيد النتائج بعمل دراسات إحصائية لها‪.‬‬
‫نستخلص‪ :‬من هذه الدراسة أن الضغط الناتج من الزحام الشديد يؤذى قاع المعدة ولكن زيادة نسبة الردة فى الطعام يقى المعدة‬
‫من هذه التأثيرات‪ .‬ولذلك فننصح بزيادة األلياف فى الطعام خاصة فى المدن شديدة الزحام‪.‬‬
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