الفهرس 
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Abstract
The respiratory system is divided into two sections; conducting
section (nose to bronchioles) form a path for conduction of the inhaled
gases and respiratory section (from respiratory bronchioles to alveoli)
where the gas exchange takes place. Lung alveoli are the functional unit
of respiratory system and each of them measuring 200 micrometer in
diameter. They are composed of type I and type II pneumocytes with
scanty amount of connective tissue inbetween. Air space of two adjacent
alveoli communicate through an alveolar pore. The interalveolar septa
inbetween alveoli have an extensive capillary bed.
Aluminum is the 3rd metal in the earth that naturally presents in
water (e.g., ponds, lakes and streams). Human is exposed to a little
amount of AL from drinking water. In the industry, the aluminum
powders are used in paints and pigments, and insert in fuel industry. In
medical part, Aluminum is used in pharmaceutical and personal care
product, in pharmaceutical industry many medications containing
aluminum as topical solutions or as drugs such as aspirin, analgesic,
antipyretic, and antacids. Aluminum is also found in infant formulae
human and cow milk, in addition, dentists use aluminum silicate as
component of dental cement.
Lastly, the aluminum chloride enters in food preservatives,
cosmetics and rubber industry as lubricants.
Propolis is a resinous wax-like bee product collected by honey
bees from plant exudates and also known as bee glue. Propolis has been
used as antioxidant and immune-stimulating. Several studies point to the
fact that propolis may be effective against environmental pollutes like
Lead. In addition, propolis has also been found to have powerful antiSummary
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inflammatory properties and can counteract the damaging effects of
aluminum. Moreover, propolis is the focus of a large number of research
projects. Propolis can modulate cytokine secretion and inhibit reactive
oxygen species (ROS). Also, it modulates the metabolism of blood lipid
leads to decrease in lipid peroxidation and scavenges the free radicals
The present study was conducted for microscopical study of
histological, immunohistochemical and morphometric changes in rat lung
following aluminum chloride administration and the possible protective
effect of propolis, regarding to its antioxidant and anti-inflammatory
activity
Materials and Methods
Fifty adult male albino rats weighing nearly 165-180 gms used in
this study. Animals were randomly divided into 5 groups:
group I (control group):-
It was composed of 10 rats, each received 2ml distilled water by
oral route and kept without any treatment for 6 weeks.
group II (Propolis supplemented):
It was composed of 10 rats, each received Propolis at a dose of
50mg/kg b.w/ day. Each rat received 1 ml distilled water containing 10
mg Propolis by oral route for 6 weeks.
group III (Aluminum chloride treated):
It was composed of 10 rats, each treated with Aluminum chloride
at dose of 475mg/kg B.W once daily, by oral gavage for 6 weeks. Each
rat received 1 ml distilled water containing 95 mg Aluminum chloride.
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group IV (Recovery group):
It was composed of 10 rats; each treated with Aluminum chloride
at adose of 95mg/kg b.w/ day for 6 weeks and then were kept untreated
for another 2 weeks.
group V (Propolis and Aluminum chloride):
It was composed of 10 rats, each received both Aluminum chloride
and Propolis once daily, by oral gavage for 6 weeks. The doses were
similar to that of the previous experimental groups.
At the end of the period of the experiment, animals were weighted
then sacrified. The lung of all groups were dissected out and their
weights were recorded. The tissues were prepared for the following
studies:
1. Light microscopical study: using haematoxylin & eosin stain,
Mallory trichrome stain, Toulidine blue stain and BCL2
immunostaining.
2. Electron Microscopic (EM) Study.
3. Morphometric study and statistical analysis.
All measurements were applied on ten non overlapping sections of
each group in the same magnification.
It was performed on the following parameters:
1) The number of pneumocytes type II (× 400);
2) the thickness of inter-alveolar septa (× 200);
3) Percentage area of collagen deposition (× 200);
4) The number of mast cells (×400)
5) The color intensity of Bcl2 immunostaining (× 400).
The calculated data were used for comparison and statistical
analysis.
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Data were statistically analyzed by SPSS. Data were expressed as
mean and SD (standard deviation).
Results
The following data have been observed in the present study:
I- General results:
By observations throughout the experiment, animals in control
group and propolis supplemented group were in a good general
condition and showed a normal behaviour, activity, eating and growth.
The mortality rate was 3. On the other hand,those in aluminum chloride
treated group showed decreased activity and appetite. But, those in
recovery group showed little changes in comparison to those in
aluminum chloride treated group. Animals in propolis & aluminum
chloride treated group showed marked improvement in their activity
and appetite.
II- MACROSCOPIC EXAMINATION:
Lungs obtained from control and Propolis groups were
homogeneous and glistening with bright red color. But, lungs of
Aluminum chloride group appeared shrunken and collapsed with dark
opaque red color and patches of hemorrhage. While lungs of recovery
group showed little hemorrhage. The lunges restored its bright red color
in the Propolis with Aluminum chloride treated group.
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III- Microscopic examination:
Light microscopic results:
A) Haematoxylin & Eosin:
group I & II:
Lung sections collected from the control and Propolis groups
(Groups I and II) showed normal lung tissue with numerous patent
alveoli. Some alveoli collected forming alveolar sacs. Each alveolus was
lined by 2 types of cells: pneumocytes I and pneumocytes II.
Pneumocytes I appeared flattened with flat nucleus and scanty cytoplasm.
Pneumocytes II appeared cubical with rounded nuclei and abundant
cytoplasm. Moreover, alveolar macrophages appeared inside the alveoli.
Thin inter-alveolar septa, blood vessels and normal bronchioles
were noticed. The bronchioles were lined by ciliated cuboidal epithelium.
group III (Alcl3 treated group):-
Aluminum chloride group revealed distorted appearance of lung
tissues. Some alveoli were collapsed and the others showed compensatory
dilatation with rupture of some inter-alveolar septa. Lung tissue had
numerous depositions of hemosiderin granules that appeared as dark
brown particles. Pnemocytes type I & II showed pyknotic nuclei, others
showed vacuolated cytoplasm with degeneration of the alveolar wall.
There was thickening of some inter-alveolar septa. The thickened
interstitium contained numerous inflammatory infiltrate and acidophilic
hyaline degeneration. The blood vessels were congested and surrounded
by inflammatory cells. There was an irregularity in the wall of the
bronchioles with sloughing of the bronchial epithelial lining. Moreover,
some hemorrhage was observed inside the bronchioles.
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group IV(recovery group):
Lung tissues showed moderate improvement after stopping
Aluminum chloride for 2 weeks where, some alveoli appeared collapsed
with pyknosis of pnemocytes nuclei, congestion of the blood vessels and
minimal cellular infiltrations were noticed. The inter-alveolar septa was
still thickened.
group V (propolis & Alcl3 treated group):-.
Co-administration of propolis with aluminum chloride showed
marked improvement in the lung tissues, where, the integrity of lung
alveoli, blood vessels and bronchioles was preserved to be more or less as
control group.
B) Mallory’s trichrome staining:
group I & II(control and propolis groups):-
Mallory’s trichrome stained sections showed few collagen fibers in
the inter-alveolar septa, around the bronchioles and around the blood
vessels.
group III (Alcl3 treated group):-
There was massive deposition of collagen fibers mainly around
bronchioles and congested blood vessels and in the inter-alveolar septa.
group IV(recovery group):
Lung sections obtained after stopping aluminum chloride for 2
weeks revealed moderate deposition of collagen fibers.
group V (propolis & Alcl3 treated group):-.
Propolis and aluminum chloride group showed minimal
collagen fibers in the inter-alveolar septa slightly more than the control
group.
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C) Toluidine blue staining:
group I & II(control and propolis groups):-
No mast cells could be detected in the control and Propolis
groups.
group III (Alcl3 treated group):-
There was dense accumulation of mast cells with rupture of some
of them.
group IV(recovery group):-
There was few number of intact mast cells in the recovery group.
group V (propolis & Alcl3 treated group):-
No mast cells were seen in this group.
D) Immunohistochemical study:-
group I & II(control and propolis groups):-
Sections of these groups showed positive brown cytoplasmic
immune reaction for BCL2 in pneumocyte type I and II.
group III (Alcl3 treated group):-
The reaction became very weak for BCL2 .
group IV(recovery group):-
Sections of this group showed moderate immune reaction to BCL2.
group V (propolis & Alcl3 treated group):-
The reaction was intense positive as the control.
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II- Electron microscopic results:
group I &II (Control and propolis groups):
Ultrathin sections of the lung in the control group showed
pneumocytes type I containing large oval nuclei with peripheral
condensed chromatin and thin attenuated cytoplasm . Pneumocytes type
II appeared rounded with large central rounded vesicular nuclei and
prominent nucleoli. Their cytoplasm contained mitochondria, lamellar
bodies, rough endoplasmic reticulum and lysosomes. The surface of
pneumocytes type II exhibited numerous short apical microvilli. The
alveolar macrophages was noticed with its characteristic nucleus and
multiple pseudopodia on the surface. The inter-alveolar septa appeared
intact with normal thickness. Ultrathin sections of Propolis group
revealed similar structure to that of the control group.
group III (Alcl3 treated group):-
Aluminum chloride group exhibited marked ultrastructure
changes where, pneumocytes type I showed flattened irregular nuclei,
swollen mitochondria and multiple cytoplasmic vacuolations. Distorted
tight junctions between the pneumocytes type I was noticed. The alveolar
macrophages were filled with numerous residual and multivesicular
bodies. Pneumocytes type II showed irregular indented nuclei. Their
cytoplasm contained multiple cytoplasmic vacuolations, distended
irregular mitochondria and dilated rough endoplasmic reticulum. The
lamellar bodies revealed degenerative changes leaving irregular empty
vacuoles. Few short microvilli appeared on the surface of pneumocytes
type II. The inter-alveolar septa contained congested blood capillaries,
degenerated pneumocytes type II, macrophages, red blood cells,
eosinophils and marked collagen fibers.
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group IV (recovery group):-
Stopping aluminum chloride treatment for 2 weeks revealed
persistance of some structural changes of the alveoli. Pneumocytes type II
showed irregular nuclei, their cytoplasm contained enlarged
mitochondria, dilated rough endoplasmic reticulum and cytoplasmic
vacuolations. Irregular lamellar bodies and disturbed microvillous border
were observed. The inter-alveolar septa still contained numerous collagen
fibers and congested blood capillaries.
group V (propolis& Alcl3 treated group):-
Sections of this group showed great reduction in the structural
changes demonstrated in Aluminum chloride group. Pneumocytes type II
restored their normal feature and appeared with large rounded vesicular
nuclei and prominent nucleoli. Their cytoplasm demonstrated numerous
rough endoplasmic reticulum , mitochondria and remarkable well defined
lamellar bodies. Some cytoplasmic vacuolations were still appeared. Mild
capillary congestion and few collagen fibers were noticed in the interalveolar
septa.
III-Statistical and morphometric results:
The animal body weight, lung weight and the ratio between them:
Aluminum chloride group revealed a highly significant decrease of
thes parameters (P< 0.001) compared to control. Recovery group showed
a significant decrease compared with control group (P≤ 0.05). There were
non-significant relation in both Propolis supplemented & Propolis and
aluminum chloride groups compared to control group.
The number of pneumocytes type II:
There was a highly significant increase in the number of
pneumocytes type II (P < 0.001) in aluminum chloride group compared to
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control. In recovery group there was a significant increase in
pneumocytes type II number compared with control group (P< 0.05)
However, However, Propolis supplemented group & Propolis and
aluminum chloride group showed a non-significant change (P>0.05)
compared to control group.
The thickness of inter-alveolar septa:
The mean thickness of inter-alveolar septa showed a highly
significant increase (P< 0.001) in aluminum chloride group compared to
control group. Recovery group exhibited a significant increase (P<0.05)
compared with control group. However, Propolis supplemented group &
Propolis and aluminum chloride group showed a non-significant change
(P>0.05) compared to control group.
The percentage area of collagen deposition:
The mean percentage area of collagen fibers showed a highly
significant increase (P< 0.001) in aluminum chloride group and a
significant increase (P< 0.05) in the recovery group compared to control.
However, Propolis supplemented group & Propolis and aluminum
chloride group showed a non-significant change (P>0.05) compared to
control group .
The number of mast cells:
The mean number of mast cells revealed a highly significant
increase (P< 0.001) in aluminum chloride group and a significant increase
(P< 0.05) in the recovery group compared to control. There was a non
significant relation (P>0.05) in both Propolis supplemented group &
Propolis and aluminum chloride group compared to control group .
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The color intensity of Bcl2 immunostaing:
The color intensity of Bcl2 showed a highly significant decrease
(P< 0.001) in aluminum chloride group and a significant decrease
(P< 0.05) in the recovery group compared to control. However, Propolis
supplemented group & Propolis and aluminum chloride group showed a
non-significant change (P>0.05) compared to control group.