THE EGYPTIAN JOURNAL OF HISTOLOGY- Vol. 30, No.2. Dec, 141 - 248, 2007. (ISSN: II10 - 0559)
THE ROLE OF GREEN TEA ON STARVATION INDUCED
MORPHOLOGICAL CHANGES IN JEJUNAL VILLI OF MALE
ALBINO RAT:HISTOLOGICAL, HISTOCHEMICALAND
SCANNING ELECTRON MICROSCOPIC STUDY
GEHAN KHALAF
Histo!og}> Department. Faculty of Medicine. Ain Shams University.
Abstract
Green Tea is a naturally occurring compound. It is recognized as a very powerful an/ioxidant, anti inflammatory
and anti carcinogenic agent. The present work was clone to investigate the role of green tea administration on
morphological changes of the jejunal villi that might occur after starvation. Twenty male albino rats were used and
divided into 4 equal groups, Group I (control rats). Group II (starved rats for 7 days), Grouplll (green tea treated
rats for 7 days after 7 days of starvation) and Group IV (recovered rats that were re/'etlfor 7 days after 7 days of
starvation). Animals were sacrificed at the end of the experiment. Jejunal villi of the starved rats were apparently
thickened. There was significant increase in the mean villas thickness and a significant decrease in the mean height
of the surface columnar cells as compared to that of the control group. The core of the villi showed edema and
cellular infiltration. There was a weak PAS positive reaction at the brush border of the columnar cells and the mean
number of goblet cells/villas was significantly decreased as compared to that of the control group. With scanning
electron microscope, starved rats showed loss of the ruicrovilli at the apex ofsome of the villi. Poorly developed goblet
cells were also observed. All these changes were improved and almost returned to normal in the green tea treated
rats(grouplll). While in recovered rats(groupIV)the improvement ofstarvation induced morphological changes of the
jejunal villi was minimum. So, it was concluded that the green lea administration could improve and accelerate the
intestinal response to refeeding after starvation.
Key Words: Starvation - Green tea - Jejunum -Histochemical - Histological.
Introduction
Green tea was reported to have a protective
effect on chemically induced tumors in the
g a s t r 0 intestinal tract in rats^. Consumption of
g r e e n t e a w a s a s s o c i a t e d w i t h t h e prevention of
chronic active gastritis*7).
_
. , .
, ,
.
...
Green tea is rich in polyphenols, which
^ . ^ a
^ ^
Qf ^
ognized
as
very
compoimds
and
are
reC
p o w e r f u i antioxidants, anti inflammatory and
8
ant j carc j n0 genic agents* '.
Starvation is a well known cause of
Gastro-mtestmal abnormalities. It causes
morphological changes in the intestinal villi .
and increased bacterial infection*1). Patients
.
.. n j , . .
undergoing surgery and nutritionally depleted
patients were kept fasting and maintained on
intra-venous nutrition. These patients showed
intestinal mucosal damaged). Autopsies of
human dying of chronic starvation have
}
.
. , ,
.•
,
The aim of the present study was to
showen gastrointestinal u Iceration, gastric and
investi
the roIe of
tea c o n s u m p t i o n on
intestinal mucosal atrophy, and submucosal
t h e morpho logical changes of jejunal villi that
hemorrhage^. In laboratory animals, acute
m i g h t occur after starvation in the male albino
deprivation of food resulted in intestinal
rats
changes which included destruction of the
villus tips, Assuring of the basal portions, and
Material and Methods
4
marked shortening of the villi* ). Anti oxidants
xj i e experiment was conducted on 20 male
-rich polyphenolic fraction isolated from green
albino rats of an average weight of 180 gm.
tea possessed anti inflammatory properties in
Animals were given water ad-libitum and
experimental animals*5).
housed in adequate ventilation and temperature.
241
3(1067-2007)
242
Gehan Khalaf
They were divided into 4 equal groups:Group I (control group): It consisted of rats
that received a normal diet
Group II (starved group): It consisted of rats
that were starved for 7 days. Water was freely
available to them.
Group III (Green tea-treated group): It
consisted of rats that were starved for 7 days
with freely available water, Then rats were
refed with a normal diet and at the same time
they were given 100 mg/kg green tea extracts
orally and daily for 7 days<9)
Group IV (Recovery group): It consisted
of rats that were starved for 7 days with freely
available water. Then refed a normal diet for 7
days.
Animals were sacrificed at the end of
experiment, Jejunal specimens were obtained
10 cm distal to the pylorus.
For light microscopic study:
Parts of the specimens were fixed in 10%
formalin. Paraffin blocks were prepared, sections
were cut at 5 \u\\ thickness and were subjected
to the following staining techniques^10)
-Maematoxylin and Eosin stain (H&E).
- Periodic acid Schiff (PAS) technique.
For scanning election microscopic study:
The other parts of the specimens were fixed
in 1.5% glutraldehyde solution in phosphate
buffer and prepared for scanning electron
microscopic study, in Anatomy Department,
Faculty of medicine, Am Shams University.
For statistical studies:
Using an automatic analysis system leica
Q500 MCO analyzer in, Histology Department,
Faculty of medicine, Ain Shams University, the
following parameters were measured:
- The mean thickness of the jejunal villus
(It was measured near tips of the villi using
H&E stained sections).
- The mean height of the surface columnar
cells (using H&E stained sections).
- The mean number of goblet cells/villus
(using PAS stained sections),
Measurements were done in randomly chosen
5 fields (under X100 magnification) of 5 serial
sections obtained from 5 different animals of
the same group and statistically analyzed using
student's t -test.
Results
Control group (group I):
H&E stained sections showed finger like
jejunal villi. The villi were seen to be covered
by surface columnar cells (enterocytes) with
oval basal vesicular nuclei and acidophilic
cytoplasm. Goblet cells were seen to be present
at intervals between enterocytes (Fig. 1). The
thickness of the villus showed a mean value of
89 um (Table I, Histogram 1). The mean height
of surface columnar cells was measured to
be 27.2 urn (Table 2, Histogram 2).
PAS stained sections showed a PAS positive
brush border and numerous goblet cells (Fig. 2).
The mean number of the goblet cells/villus were
measured to be 25.6 (table 3> Histogram 3).
Scanning electron microscopic examination
of the surface of the villi of the control group
showed numerous goblet cells (Fig. 3A). The
cut surface of the villi showed regularly arranged
micro villi of the surface columnar cells (Fig. 3B).
Starved group (group II):
In H&E stained section, the core of the
villi showed edema and mononuclear cellular
infiltration. There was thickening of the villi
and an apparent decrease in the height of
the surface columnar cells with irregularly
arranged nuclei (Fig. 4A, E). These results
were confirmed by histomorphometric study
which revealed that the mean thickness of
the villi was highly significantly increased
(173 |im) (Table 15 Histogram 1) and the
mean height of the surface columnar cells
was highly significantly decreased (17.1 jim)
(Table 2, Histogram 2) as compared to those of
the control group.
PAS stained sections showed a weak or even
absence of the PAS positive reaction at the
brush border of the surface columnar cells and
few goblet cells (Fig 5). The mean number of
The Role of Green Tea on Starvation Induced Morphological Changes in Jejunal villi
the goblet cells/villus was highly significantly
decreased (5.2) (Table 3, Histogram 3) as
compared to that of the control group.
Scanning electron microscopic study showed
poorly developed goblet cells (Fig.6 A). The cut
surface of the villi showed loss of the microvilli
of the surface columnar cells at some areas
(Fig.6B).
Green tea treated group (group III):
H&E stained sections showed that the
villous structure was almost similar to that
of the control group (Fig. 7). There was non
significant change in the mean thickness of
the villi (91 urn) (Table 1, Histogram 1) and
the mean height of the surface columnar cells
(25.4um) (Table 2, Histogram 2) as compared
to those of the control group.
PAS stained sections showed a strong PAS
positive reaction at the brush border of the
surface columnar cells and numerous goblet
cells (Fig 8). The mean number of the goblet
cells/villus was non-significantly changed
(20.9) (Table 3, Histogram 3) as compared to
that of the control group.
Scanning electron microscopic study showed
the jejunal villi to be nearly similar to that of the
control group (Fig.9A). The cut surface of the
villi showed regular microvilli of the surface
columnar cells (Fig 9B).
Recovery group (groupIV):
In FI&E stained sections, some villi appeared
nearly as that of the control group but still many
other villi showed edema and cellular infiltration
in its core, thickened villi and an apparent
decrease in the height of the surface columnar
cells (Fig. 10). There was a highly significant
increase in the mean thickness of the villi
(132 urn) (Table 1, Histogram 1) and a highly
significant decrease in the mean height
of the surface columnar cells (19.3j.tm)
(Table 2, Histogram 2) as compared to
those of the control group.
In PAS reaction, there was a weak PAS
positive reaction at the brush border of the
surface columnar cells and few goblet cells
(Fig 11). The mean number of the goblet cells/
villus was highly significantly decreased (6,1)
(Table 3, Histogram 3) as compared to that of
the control group.
Scanning electron microscopic study showed
that some of the jejunal villi appeared nearly
similar to that of the control group. Most of the
villi revealed few goblet cells and loss of the
microvilli of the surface columnar cells in some
areas (Fig. 12A, B).
The mean thickness of the villus
200(
Micrometers •J50j
wo\
Table (1): showing the changes in the mean thickness of the jejunal villus
SOX
in the different groups in comparison to the control group (I).
Control (1)
Starved! II)
Green lea
trealed(lll)
Recovered! IV)
Mean.
urn
S.D
89
173
91
132
10.1
25.3
9.01
14.3
T
6.895
0.322
5.492
P
<0.001
>0.05
<0.001
243
ll
111
Groups
Significance
HS
NS
HS
IV
Histogram (I): showing the changes in the mean thickness of
the jejunal villus in the different groups in comparison to
the control group (1),
The mean height of the surface columnar cells
Table (2): showing the changes in the mean height of the surface columnar
cells in the different groups in comparison to the control group (f).
Group
Mean.
urn
S.D
T
Control (1)
Starved! 11)
Green lea
trcalcd(lll)
Recovered! IV)
27.2
17.1
25.4
19.3
2.6
1.16
3.8
1.02
7.94
0.875
6.325
P
<0.001
>0.05
<0.001
Micrometers
Significance
HS
NS
HS
Histogram (2): showing the changes in the mean height of the
surface columnar cells in the different groups in comparison
to the control group (I).
Gehan Khalaf
244
Table (3): showing the changes in the mean number of the goblet cells
villus in the different groups in comparison to the control group (I).
Group
Control (I)
Starvcd(ll)
Green lea
trcated(lll)
Rccovcrcd(IV)
Mean.
u.m
25.6
5.2
20.9
6.1
S.D
8.9
1.04
7.2
1.01
T
I'
The mean number of goblet cell
/villus
Significance
Micrometers
5.0912
0.918
4.9
<0.00l
>0.05
<0.001
IIS
NS
IIS
30
20
10
0
II
III IV
Groups
Histogram (2): showing the changes in the mean height of the
surface columnar cells in the different groups in comparison
to the control group (I).
—
WLM
' 4
%
*
?.« °t>
m
%.
:
ig. {I): Shows finger like jejuna! villi. The villi are covered with
tall columnar cells (enlerocytes) (e) and goblet cells (g).
Control group (I). Kf&E X250.
»»
V
ig. (2): Shows strong PAS positive reaction (j) at the brush
border of the columnar epithelial cells covering the villi.
Numerous goblet cells (g) are seen.
Control group (I). PAS X400.
Fig. (3): A- Shows the numerous goblet cells (g) on the surface of the villi. B- The cut surface of the villous shows the reaular
appearance of the microvilli (|),
: ___
Control group (I). SEM. AX I O0O.BX200O.
The Role of Green Tea on Starvation Induced Morphological Changes in Jejunal villi
245
*.•
_1
t
Fig. (4): A-Sho\vs an apparent increase in the thickness ofthe villi, edema (d) .cellular infiltration (f) in its core. B-Thc surface
columnar epithelial cells show an apparent decease in height with irregularly arranged nuclci(j).
Starving group (II).I l&E AX250 BX640.
Fig. (5): .Shows a weak PAS- positive reaction (1) at the
brush border of some ofthe columnar epithelial cells
covering the villi. There is a very few number of the
goblet cells(g).
Starving group (II). PAS X400.
lux V *,pol M.iqn
?bOVV3fc bbOOx
llol
'.I
WO I Xf>
88 0
l)H IIAMIJ'f Al I
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dtak
Fig. (6): A- Shows poorly developed goblet cells. B- The cut surface ofthe villous shows loss ofthe microvilli insome areas (f),
Starving group (II) SEM. AX 1200,13X6500..
Fig.(7): Shows that the villous structure is almost similar
to that ofthe control group.
Green tea -treated group (III).H&E X250.
Fig. (8): Shows an almost normal appearance ofthe PAS
positive reaction of the goblet cells and the brush
border of columnar epithelial cells covering the villi.
Green tea- treated group (lll).PAS X400.
Gehan Khalaf
246
B
ccV SpolMagn
260kV36 6000X
wm
Dot WD Exp
SE 9.8 0
DRHAMDYALY
1g. {{)y. A- Shows numerous goblet cells (gJ.B- The cut surface of the villous shows regular inicrovilii (|).
Green tea-treated group (III)..SEM.AX]000,BX5000.
i
Y
d
■ 1
!!■ *J?
* ft "» *
■*•. *k
»>
k/
W ' ^ .
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Fig.(IO): Shows an apparently thickened villi and edema
(d) and cellular infiltration (f) in its core.
Recovery group (IV).l I&E 250
ig. (11): Shows a weak PAS positive reaction (T) at the brush
border of the columnar epithelial cells and a few numbers
of the goblet cells (g).
Recovery group (IV).PAS X400
tVX V Spol M.iqn
? 6 0 k V 3 6 bOOx
Dot WD [ xp
fit
04 0
Fig. (12): A- The surface of the villous shows poorly developed goblet celts. B- The cut surface of the villous shows loss of the
inicrovilii in some areas (T).
Recovery group (IV). SEM. AX500.BX3500.
The Role of Green Tea on Siarvaiion Induced Morphological Changes in Jejanal villi
Discussion
In the present study, starvation for 7 days
(group II) resulted in profound morphological
changes of the jejunal villi. There were edema
and cellular infiltration in the core of the villi.
The mean villus thickness was highly significant
increased as compared to that of the control
group. The surface columnar epithelial cells
showed irregularly arranged nuclei, absence
of PAS positive reaction at their brush border
and a highly significant decrease in their mean
height as compared to the control group. These
■ morphological changes were also demonstrated
by previous authors*11). They studied jejunal
mucosa in infantile marasmic malnutrition and
found broadening and irregularity of the villi,
a decrease in the epithelial height, irregular
nuclear arrangement* thin brush border and an
increased number of lymphocytes and plasma
cells in the lamina propria. Cellular infiltration
in the core of the villi that was seen in the present
work was explained by some authors*1'2> who
reported that in experimental animals nutrition
depletion led to increase bacterial infection. In
the current study scanning electron microscopic
examination of the villi of the starved rats
revealed loss of the microvilli in some areas. It
was also reported that food deprivation induced
a marked decrease in the brush border enzymatic
activity*1-). Starvation, in the present study,
resulted in a highly significant decrease of the
mean number of the goblet cells as compared
to that of the control group. This intestinal
damage that was induced by starvation was
explained by many authors. It was found that
food deprivation produced a reduction in the
cell renewal, impaired cell differentiation and
reduced the rate of migration of epithelial cells
to the villous tips*4*. This intestinal damage was
attributed to increase super-oxide radicals in the
villi and the crypts*13).
The present study showed that green tea
administration improved the small intestinal
repair after starvation induced damage. In the
green tea-treated rats (groupIII), the villi were
nearly similar to those of the control group.
There was a strong PAS positive reaction at the
247
brush border of the columnar epithelial cells
and normal appearance of the goblet cells. SEM
revealed an almost normal structure in most of
the villi. It was previously explained that the
free oxidants radicals could elicit epithelial
cells necrosis and death and they were reduced
by addition of dietary antioxidants*1^. It was
also reported that green tea polyphenols had
potent antioxidant properties*153. It scavenges
the toxic oxidants and blocks the activation
of the transcription factor, "NF-B that controls
expression of genes active in inflammation
including cytokines e.g IL, TNF , enzymes eg.
nitric oxide synthasc*16), It was observed that
these inflammatory products were associated
with gastritis and epithelial cell death*17*.
Other workers reported that green tea and its
constituent polyphenols were associated with
reduced expression of inflammatory genes
responsible for its anti-inflammatory effects*1*),
Previous studies revealed that green tea
administration reduced the severity of colitis
in mice which was in the form of epithelial
hyperplasia, mononuclear cell filtration,
ulceration and few goblet cells*19*. Green tea
was widely distributed throughout the body
and its highest concentration was found in the
intestine*20). This might explain the beneficial
effect of the green tea on the starvation induced
intestinal damage that was observed in the
present study.
In die recovered rats (group IV), some villi
appeared to be normal but many others showed
edema and cellular infiltration in the core of the
villi. Weak PAS positive reaction at the brush
border of the columnar epithelial cells and few
goblet cells were observed. It was reported that
during nutrition rehabilitation of marasmic
infants, there was no reversal in architecture
of the intestinal mucosa, although its thickness
approach normal and it was not known for how
long marasmic infants need to be maintained
on adequate diet before the microscopical
appearance becomes entirely normal*11), So it
was concluded that green tea administration
could improve and accelerate the intestinal
response to refeeding after food deprivation,
Gehan Khalaf
248
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