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The possible protective role of bone marrow transplantation on irradiated mothers and their fetuses

Authors:
  • Jouf University

Abstract

This work was conducted to evaluate the possible protective role of bone marrow transplantation (BMT) against whole body γ-irradiation (2Gy) in pregnant albino rats and their fetuses at two different gestation periods. Different treatments were performed on days 7or 14 of gestation and examined at the end of the gestation period (day 20).Pregnant rats irradiated at 2Gy γ-rays on day 7 or 14 of gestation showed unequal distribution of implantation sites between the two horns, reduction in the number of implantation sites and one case of complete abortion on day 7 of gestation, but on day 14 of gestation, there were lots of resorbed embryos. Fetuses showed very thin skin layers, subcutaneous hemorrhage, severe growth retardation and malformed rostrum, eyes and eye lids in addition to malformed fore and hind limbs and tails. Bone marrow transplantation showed no detectable changes in morphology of the fetuses. Also, radiation caused many histological and histochemical changes in the lung tissue of mothers and their fetuses on day 7 or 14 of gestation, but bone marrow transplantation post-irradiation highly improved the histological and histochemical architecture of the liver tissues.
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The possible protective role of bone marrow transplantation on irradiated mothers and their fetuses
Nehal A. Abuo El Naga and Mervat A. Abd Rabou
Zoology Department, Faculty of Science, Al- Azhar University; Egypt
Nehal.61@hotmail.com
Abstract: This work was conducted to evaluate the possible protective role of bone marrow transplantation (BMT)
against whole body γ-irradiation (2Gy) in pregnant albino rats and their fetuses at two different gestation periods.
Different treatments were performed on days 7or 14 of gestation and examined at the end of the gestation period
(day 20).Pregnant rats irradiated at 2Gy γ-rays on day 7 or 14 of gestation showed unequal distribution of
implantation sites between the two horns, reduction in the number of implantation sites and one case of complete
abortion on day 7 of gestation, but on day 14 of gestation, there were lots of resorbed embryos. Fetuses showed very
thin skin layers, subcutaneous hemorrhage, severe growth retardation and malformed rostrum, eyes and eye lids in
addition to malformed fore and hind limbs and tails. Bone marrow transplantation showed no detectable changes in
morphology of the fetuses. Also, radiation caused many histological and histochemical changes in the lung tissue of
mothers and their fetuses on day 7 or 14 of gestation, but bone marrow transplantation post-irradiation highly
improved the histological and histochemical architecture of the liver tissues.
[Nehal A. Abuo El Naga and Mervat A. Abd Rabou. The possible protective role of bone marrow
transplantation on irradiated mothers and their fetuses. Stem Cell 2012;3(3):8-30] (ISSN 1545-4570).
http://www.sciencepub.net. 2
Keywords: bone marrow transplantation (BMT); γ-irradiation (2Gy); pregnant albino; fetus
1. Introduction:
The damaging effects of ionizing radiation
lead to cell death and are associated with increased
risk for number of diseases (Halliwell and Aruoma,
1991).
Gamma-rays caused high incidence of
intrauterine mortality, resorption of embryos and
inhibition of gestation to pregnant rats (El-Naggar et
al., 1996).
The development of new concerns comprises
immune function and / or radiation induced genetic
damage (Trosko, 1996).
Leadon (1996) stated that ionizing radiation has
been considered as a source for causing physical
damage to living organisms. Exposure to ionizing
radiation eventually results in injuries to the
biological system depending on the dose, duration
and type of radiation exposure besides the radio-
sensitivity of different tissues (Pecaut et al., 2001).
Ionizing radiation forms radicals in the DNA and in
the surrounding water molecules of the hydration
shell of the DNA, which in turn destroy DNA
(Kopjar et al., 2006; Moss, 2012).
De Santis et al. (2005) suggested that ionizing
radiation represented a possible teratogen for the
fetus, but this risk has been found to be dependent on
the dosage and the effects correlatable to the
gestation age at exposure.
Embryonic death occurred at the early stages of
gestation by ionizing radiation (Devi and Hande,
1990).
Abu Gabal et al. (1994a) mentioned that 2Gy
gamma rays provided to rats at day 4 of gestation
caused prenatal death beside a small percentage of
resorbing bodies, subcutaneous haemorrhage and
diminution in size with clubbed fore and hind limbs.
Salama (2004) estimated abnormal implantation sites
when pregnant rats were exposed to whole body
gamma rays (1Gy/3 times) on the 7th, 11th and 15th
days of gestation.
Ramadan (2007a) exposed pregnant rats to
whole body gamma rays at a dose of 0.5Gy for 4
times on gestational days 9, 10, 11 and 12, she
detected a significant decrease in fetal numbers. She
added that radiation exposure induced malformations
including excencephaly, diminution of size and
kypophyis.
Bone marrow is a complex tissue composed of
two compartments, haematopoietic and stromal one.
The stromal compartment is the structural basis of the
haematopoiteic microenvironment which is a
complex tissue that contains a subset of cells termed
mesenchymal stem cells (MSCs ) (Caplan , 1991).
Bone marrow transplantation at different time
periods after radiation exposure induces its
restorative effect when the treatment is done post
irradiation asconfirmed by raiozinc uptake (Kafafy ,
1993)
Abu- Sinna et al. (2005) cited that BMT is
known to cause regeneration of thymus, spleen and
bone marrow after lethal whole body irradiation.
Bone marrow transplantation to pregnant mice post-
exposure to γ-rays improved the developmental and
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structural changes in the fetus (Wang, 2001;
Mansour, 2012)
The lung of fetuses obtained from mothers
exposed to 3Gy on day 6 of gestation showed severe
degenerated alveolar cells, narrow alveolar intercepts
with presence of many pyknotic nuclei (Abu El
Naga, 1989).
Lung damage post-irradiation was also detected
by Liao et al. (2000) in mice exposed to single doses
of X-rays ranging from 12 to 20 Gy. They observed
increased morbidity from radiation pneumonitis and
lethality between 12 to 32 weeks after irradiation.
They also noticed many histological changes in the
lung tissue.
Radiation pneumonitis was observed in lung of
mice exposed to radiation (Yan et al., 2004) and in
Brown Norway rat (Eveline et al., 2009). They also
stated that radiation exposure with a high dose rate
(0.8Gy/ min) or low dose rate (0.05 Gy/ min) caused
many pathological changes in lung of rats such as
severe aplasia of hemopoietic and lymphoid tissues
with increased hemorrhagic areas.
El-Khatib et al. (2009) irradiated mice with
X-rays doses of 5 to 14Gy for sex weeks. They
noticed that mice irradiated with 5 and 7Gy exhibited
no changes in lung density, but those exposed to
doses greater than 10Gy exhibited marked increases
in lung density. They also noticed pneumonitis in the
lungs of exposed mice.
In 2010, Kirsch et al., noticed increased lung
cancer in mice post-irradiation (15.5Gy).
The present study aimed to evaluate the possible
protective effect of BMT against radiation injury in
pregnant rats and their fetuses.
2. Material and Methods
A. Experimental animals:
Mature albino rats (Rattus albinus) ranging
from 120-150 gm body weight were housed in cages,
six females per cage. The males were kept separated
from females until mating. Females of proestrous and
estrous periods were housed with males (2:1).
All rats were kept under normal conditions
and fed pellets concentrated diet and vitamin
mixtures.
Pregnancy was assured next morning by the
presence of vaginal plug. The presence of
spermatozoa in smears of vaginal content confirmed
that mating had taken place and that day was taken as
the first day of pregnancy.
B. Radiation facility:
Irradiation was performed by Gamma-cell 40 (137
Cesium) belonging to the National Center for
Radiation Research and Technology "NCRRT",
Atomic Energy Authority, Cairo, Egypt. The 137
Cesium source activity provided a dose rate
0.48Gy/min.
C. Bone marrow transplantation:
Bone marrow transplantation (BMT) donors
and recipients were chosen of the same inbred strain.
Sisters to sisters (syngenic transplantation). The
donors were sacrificed by cervical dislocation and
femur bones were cleaned and both ends were
chipped by bone nibbling forceps. The marrow was
blown of the femur into saline solution under
sterilized conditions surrounded by ice cubes, and
mixed by drawing and expelling it several times from
the syringe without needle in order to avoid
mechanical damage to the cells. Total viable of cells
about 75 x 106 ± 5 were injected one hour post
irradiation (Decleave et al., 1972).
D. Experimental design:
Pregnant animals were divided into the following
groups (6 females each):
1- Normal untreated control pregnant rats (C). 2-
A group of pregnant rats exposed to 2Gy on day 7 of
gestation (R7). 3- A group of pregnant rats exposed to
2Gy on day 7 of gestation and received freshly drawn
BMT (75 × 106 ± 5 cells) by i.p. injection 1 h post-
irradiation (R7+BM). 4- A group of pregnant rats
exposed to 2Gy on day 14 of gestation (R14). 5- A
group of pregnant rats exposed to 2Gy on day 14 of
gestation and received one dose of (BMT) by i.p.
injection 1 h post-irradiation (R14+BM). All animals
were sacrificed on day 20 of gestation.
E. The morphological study:
The pregnant females were dissected and the
uterine horns were removed and immediately
photographed. The uterine form and resorptions,
fetuses malformations were carefully examined
grossly for anatomical abnormality in rostrum, eye,
digits, limbs and tails.
F. The histological and histochemical studies:
On day 20 of gestation, pregnant rats were
sacrificed, small pieces of mothers and fetal’s lung
tissues were quickly removed and fixed in 10%
neutral buffer formol and Carnoy’s fluid for the
histological and histochemical studies. Specimens
were washed and dehydrated in ascending grades of
alcohol, cleared in xylene and embedded in paraffin
wax. Sections were then cut at 5µ thickness and
stained by haematoxylin and eosin stain according to
the method of Drury and Wallington (1980),
Mallory’s trichrome stain for demonstrating collagen
fibers (Pearse, 1977), periodic acid Schiff΄s
technique for demonstrating polysaccharides in the
lung (Pearse, 1977), mercuric bromophenol blue
method for detecting total protein (Mazia et al.,
1953).
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3. Results:
Morphological observations:
Anatomical observation of the uteri of the
control rats showed healthy bright appearance and
normal distribution of implanted fetuses between the
two horns (plate 1A)
Irradiated pregnant rats on the 7th day of
gestation showed reduction in the number of
implantation sites (Plate1B), but R14 group showed
resorption of most fetuses which gave the uterus a
dark color (Plate1D) with reduction in number of
fetuses (Plate1E).
Animals irradiated at 2Gy on the 7th day of
gestation and intraperitonealy injected with BM cells
1 h post-irradiation revealed normal distribution of
implantation sites in the two horns of the uterus
(Plate 1C). A slight improvement in the number and
distribution of implantation sites was noticed in
R14+BM group(Plate 1F).
Plate (1): photomicrographs of uteri of rats sacrificed on day 20 of gestation showing: A; control group, B;
reduction in the number of implantation sites(R7 group), C; normal distribution of implantation sites (R7+BM group),
D&E (R14 group); resorption of most fetuses which gave the uterus a dark color with reduction in number of
implantation sites respectively, E; a slight improvement in the number and distribution of implantation
sites(R14+BMgroup).
A
B
C
D
E
(A
(B
(C
(D
(E
(F
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Normal control fetuses taken on the 20th day
of gestation showed normal size and length (Plate
2A). Reduction in the size of fetuses of R7 group was
recorded with very thin skin layers (Plate 2B).
Fetuses of R14 group showed subcutaneous
haemorrhage in the neck region, dead fetuses, growth
retardation, reduction in length, kyphosis and
excencephaly (plate 2D&E).
Animals of group R7+BM showed that size and
length of fetuses were near to the control group
(Plate 2C). A slight improvement in size and length
of fetuses was recorded in fetuses of group R14+BM
(Plate 2F).
Plate (2): Photomicrographs of fetuses of rats sacrificed on day 20 of gestation showing: A; control group, B,
(R7 group); reduction in the size of fetuses with very thin skin layers, C; improvement in the size and length
compared with the control one (R7+BM group), D&E,(R14 group);verythin skin and reduction in length in all fetuses,
subcutaneous haemorrhage in the neck region (↑), dead fetus (↕), kyphosis (▲) and exencephaly (↑↑) ,E; a slight
improvement in the size and length of fetuses (R14.+BMgroup).
A
B
C
D
E
G
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Normal upper and lower jaws were recorded
in the fetuses of R7 and R7+BM groups compared with
the control fetuses (Plate 3A), but, unequal jaws
were detected in all fetuses of R14 group and some
fetuses of R14+BM group (Plate 3B).
Normal eye lids and protrusion of eye ball in
fetuses of the control group (Plate 3C). Also, this
was observed in fetuses of groups R7+BM& R14.
Fetuses of group R7 were devoid of eye lids with
exophathalamus (Plate 3D). No protrusion of eye
ball was noticed in fetuses of R14+BM group (Plate
3E).
Plate (3): photographs of the rostrum and eye regions of 20-day old fetuses showing: A; normal upper and
lower jaws (control group), B; unequal jaws (R14 or R14+BM groups), C; presence of eye lids (↑) (control group), D;
devoid of eye lids with exophathalamus (↑) (R7 group), E; no protrusion of the eye ball (↑) (R14+BM group).
B
A
E
D
C
A
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Fetuses of the control group showed presence
of five digits or toes in the fore and hind limbs
(plates 4A&5A). In fetuses of R7 group, malformed
fore limbs were observed. Digits were smaller than
the control with presence of syndactly in the other
cases (plate 4B&C). Adactly was observed in most
fetuses of R14 group (Plate 4D). No malformations
or change in the number of toes were noticed in the
fore limbs of the fetuses in R7+BM& R14+BM groups
when compared to fetuses of the control group.
Ectrodactly and adactly were detected in fetuses of R7
group (Plate 5B&C). All fetuses of R14 group and
some fetuses of R14+BM group showed presence of
syndactly or meromelia (Plate 5D, E& F).
Normal tails were detected in fetuses of the
control group and R7+BM groups (Plate 6A). In some
cases of each treated groups, the tail was malformed
such as twisted and short& thin tail in fetuses of R7
group (Plate 6B, C&D). Also, the short and thin tail
was observed in fetuses of R14 group (Plate 6E). One
case of reversed tail was found in a fetus of group
R14+BM (Plate 6F).
Plate (4): photographs of the fore limb region of 20-day old fetuses showing: A; presence of five toes (control
group), B; digits are smaller than the control (R7 group), C&D (R14 group); presence of syndactly and adactly
respectively.
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Plate (5): photographs of the hind limb region of 20-day old fetuses showing: A; presence of
5 toes (control group), B, C (R7 group); presence of ectrodactly and adactly respectively, D, E&F
(R14 and R14+BM groups); presence of syndactly or meromelia.
A
B
C
D
E
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Plate (6): photographs of the tail region of 20 day old fetuses showing: A; control group, B, C&D (R7 group);
twisted tail, short and thin tail respectively, E; short and thin tail (R14 group), F; reversed tail (R14+BM group).
B
D
A
C
E
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Fig.(1):showing lung tissue of a pregnant control rat. Bronchiole(b), alveolar sac(as),alveolar septa (↑). (H &E x 100)
Fig.(2): showing lung tissue of a pregnant rat of R7 group. Notice: areas of granuloma cells (g), highly congested and dilated
artery (a) and congested alveolar septae (↑). (H &E x 100)
Fig.(3): showing somewhat lung tissue of pregnant rat of R7+BM group, but, some alveolar septae were thickened. (H &E x 100)
Fig.(4):showing lung tissue of a pregnant rat of R14 group. Notice: highly thickened, corrugated and distorted arterial wall. Most
nuclei of cells of the lung tissue appeared (H &E x 100)
Fig.(5): showing lung tissue of a pregnant rat of R14+BM group. Some alveolar septae were highly thickened with numerous
pyknotic nuclei in their epithelial cells and cells of the bronchiole. Nearly the lumen of the artery was oblitrated due to the
thickened arterial wall. (H &E x 100)
2
4
1
3
5
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Fig.(6): showing normal distribution of the collagen fibers in the lung tissue of a control pregnant rat. (Mallory’s trichrome
stain x 100)
Figs.(7):showing lung tissue of a pregnant rat of R7 group. Notice: highly fibrotic walls of the bronchioles, arteries and veins
with highly increased collagen fibers in the thickened alveolar septae and around the walls of the bronchioles. (Mallory’s
trichrome stain x 100)
Fig.(8):showing normal distribution of collagen fibers in the lung tissue of a pregnant rat exposed of R7+BM group. (Mallory’s
trichrome stain x 100)
Figs.(9): showing collagen distribution in the lung tissue of a pregnant rat exposed of R14 group. Notice: fibrotic walls of the
arteries and veins with highly increased collagen fibers in the distorted walls of the bronchioles and thickened alveolar septae.
Notice also fatty degeneration (↑). (Mallory’s trichrome stain x 100)
Fig.(10): showing collagen distribution in the lung tissue of a pregnant rat of R14+BM group. Notice: numerous fibrotic areas (F)
and increased collagen fibers in the highly thickened alveolar septae. (Mallory’s trichrome stain x 100)
10
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Fig.(11): showing normal distribution of polysaccharides in the lung tissue of a control pregnant rat. (PAS X 100)
Figs.(12): showing increased stain affinity of polysaccharides in the highly thickened walls of arteries, alveolar septae and bronchioles.
Debris of the epithelial cells inside the lumen of the bronchioles acquired moderate stain affinity (↑).Granuloma cells acquired dense stain
affinity (g) in the lung tissue of R7 group. (PAS x 100)
Fig.(13): showing normal polysaccharides content in the lung tissue of a pregnant rat of R7+BM group. (PAS X 100)
Fig.(14): showing polysaccharides distribution in the lung tissue of a pregnant rat of R14 group. Notice: depleted areas of fatty
degeneration (↑), haemolysed RBCs inside the highly corrugated and elongated arterial wall appeared faintly stained, while, some
thickened alveolar walls, granuloma cells and thickened arterial walls were deeply stained. (PAS x 100)
Fig.(15): showing polysaccharides distribution in the lung tissue of a pregnant of R14+BM group. Notice that alveolar septae, arterial wall
and the fibrous layer encircling the bronchiole acquired a dense stain affinity. (PAS X 100)
11
15
12
14
13
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Fig.(16): showing normal total protein content in the lung tissue of a control pregnant rat. (Mercuric bromophenol blue x 100)
Fig.(17): showing densely stained total protein in the granuloma cells inside and outside the bronchiole (↑), the arterial wall and
also in the thickened alveolar septae in the lung tissue of a pregnant rat of R7 group (Mercuric bromophenol blue x 100)
Fig.(18): showing somewhat content of total protein in the lung tissue of a pregnant rat of R7+BM group. (Mercuric bromophenol
blue x 100)
Figs.(19): showing total protein in the lung tissue of a pregnant rat of R14 group. Notice: deeply stained arterial and bronchial
walls. Some alveolar septae appeared densely stained. (Mercuric bromophenol blue x 250)
Fig.(20): showing deeply stained total protein in the walls of bronchioles and arteries. Also some alveolar septae were densely
stained in the lung tissue of a pregnant rat of R14+BM group. (Mercuric bromophenol blue x 100)
16
20
19
18
17
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Fig.(21): showing fetal lung tissue of a control pregnant rat. (H &E x 100)
Figs.(22):showing fetal lung tissue of R7 group. Notice: highly dilated and congested blood vessels with numerous
haemorregic areas around the bronchiole and alveolar septae with presence of micronucleus (↑). Fibrosis was
detected beside the wall of bronchiole (f). (H &E x 100)
Fig.(23): showing nearly normal fetal lung tissue of R7+BM group with exception of the congested alveolar septae. (H
&E x 100)
Figs.(24):showing fetal lung tissue of R14 group. Notice: delaminated epithelial layers of the bronchioles (↑) which
lost their normal architecture and surrounded by complete fibrous layers, highly distorted arterial wall (^) which
contained haemolysed blood cells and alveolar septae lost their normal architecture. The lumen of the bronchiole
contained debris of degenerated epithelial cells.(H &E x 100)
Fig.(25): showing nearly normal bronchiole and alveolar septae in the fetal lung tissue of R14+BM group, but some
alveolar septae were congested .H &E x 100)
21
25
24
23
22
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Fig.(26): showing normal distribution of collagen fibers in the fetal lung tissue of a control pregnant rat.(Mallory’s trichrome
stain x 100)
Figs.(27): showing collagen fibers in the fetal lung tissue of R7 group. Notice: highly increased collagen fibrers in the walls of
bronchioles, dilated blood vessels, thickened alveolar septae and blood vessels. (Mallory’s trichrome stain x 100)
Fig.(28): showing collagen fibers in the fetal lung tissue of R7+BM group followed by bone marrow transplantation one hour post-
irradiation. Notice: a slight increase in collagen fibers in and around walls of bronchioles, blood vessels and alveolar septae.
(Mallory’s trichrome stain x 100)
Fig.(29):showing collagen fibers in the fetal lung tissue of R14 group. Notice: increased collagen fibers in the branched and
thickened walls of the bronchioles, fibrotic areas and thickened alveolar septae. (Mallory’s trichrome stain x 100)
Fig.(30): showing nearly normal content of collagen fibers in the fetal lung tissue of R14+BM group. (Mallory’s trichrome stain
x 100)
26
29
28
27
30
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Fig.(31):showing normal distribution of polysaccharides in the fetal lung tissue of a control pregnant rat. (PAS x100)
Figs.(32):showing polysaccharides distribution in the fetal lung tissue of R7 group. Notice: increased stain affinity of
polysaccharides in RBCs found inside the congested blood vessels and haemorrhagic areas. Fibrous layers encircling walls of the
bronchioles showed moderate stain affinity. Thickened alveolar septae showed increased stain affinity(↑). (PAS x100)
Fig.(33): showing normal distribution of polysaccharidesin the fetal lung tissue of R7+BM group. (PAS x100)
Fig.(34):showing dense stain affinity of polysaccharides in the delaminated epithelial layer of the bronchiole, thickened alveolar
septae and arterial wall of the fetal lung tissue of R14 group. (PAS x100)
Fig.(35): showing polysaccharides in the fetal lung tissue of R14+BM group. Notice: a slight increase in stain affinity of
polysaccharides in the wall of the bronchiole and alveolar septae due to increased RBCs.(PAS x100)
31
32
35
34
33
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Fig.(36): showing normal distribution of total protein in the fetal lung tissue of the control group. (Mercuric bromophenol blue
x 100)
Fig.(37):showing total protein in the fetal lung tissue of R7 group. Notice: increased stain affinity of total protein in the highly
congested and elongated arterial wall, but wall of the bronchiole and alveolar septae appeared less stained. (Mercuric
bromophenol blue x 100)
Fig.(38): showing nearly normal content of total protein in the fetal lung tissue of R7+BM group, but the congested artery
contained deeply stained RBCs. (Mercuric bromophenol blue x 100)
Fig.(39): showing total protein in the fetal lung tissue of R14 group. Notice: faintly stained alveolar septae and walls of the
bronchioles which were surrounded by densely stained fibrous layers (Mercuric bromophenol blue x 100)
Fig.(40):showing increased stain affinity of total protein in the thickened alveolar septae and the fibrous layer surrounding walls
of the bronchioles of the fetal lung tissue of R14+BM group. (Mercuric bromophenol blue x 100)
36
40
39
38
37
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4. Discussion
The biological effect of radiation correlates with
the given doses (Fowler, 1994). The most important
target in living cells is DNA (direct target theory),
but more often it indirectly damages DNA by
including the formation of free radicals, particularly
those that form the radiolysis of water (indirect target
theory). Other cell molecules that may also be direct
or indirect targets of radiant injury include lipids in
cell membranes and proteins that function as critical
enzymes. The transfer of energy to a target atom or
molecules from the incident source of radiant energy
occurs within micro fractions of a second, yet its
biological effect may become apparent for minutes
or, if the effect is on DNA, even decades (Kumar et
al., 2003).
The interaction of ionizing radiation with the
biological system resulted in generation of reactive
oxygen species (ROS) or free radicals (Gracy et al.,
1999; Strinivasan et al., 2006; Mansour, 2012).
Free radicals cause oxidative stress where
antioxidants decrease lipid peroxidation (Basaga,
1990; Karbowink and Reiter, 2000; Nordberg
and Arner, 2001).
According to Heibashy (1990) ionizing
radiation caused destructive effect on the cells of
tissues which release enzymes from organells,
moreover ionizing radiation caused alternation in the
ability of enzymes to hydrolyse phosphate esters.
Damage can occur due to direct ionization of DNA
molecule itself or indirectly through the formation of
toxic products, such as free radicals and free ions that
interact with any molecule in their path (ATSDR,
1999). Several studies have suggested that infield
DNA damage after lung irradiation is caused by both
the direct effects of radiation and the indirect effects
of the inflammatory response, whereas the out-of-
field damage may be caused by the indirect effects of
the inflammatory response alone. The exact
mechanisms involved in the inflammatory response
to radiation are unknown; however, it has been
suggested that the generation of ROS (and RNS)
immediately after irradiation, together with a cyclic
(and chronic) up-regulation of inflammatory
cytokines and the recruitment of inflammatory cells
such as macrophages and neutrophils, is responsible
for the damage seen in the lung after irradiation
(Khan et al., 2003; Fleckenstein et al., 2007)
Physiological alternations take place in the
mother during pregnancy. These changes are
profound and vital for the successful completion of
gestation. Many of these adaptations are hormonally
mediated and others are attributed to the effect of
gravid uteri (Bocking, 1994).
Exposure to gamma rays during the intrauterine
development can produce a broad spectrum of
congenital abnormalities, growth retardation,
developmental delays and functional defects
(Kiskova and Smajda, 2006).
Pompfer et al. (1992) concluded that exposure
to 2.05Gy X-rays at day 8 after conception resulted in
increasing malformations in the sensitive tissues and
caused defects in the eye, skull and nervous system
as results of delayed cell division.
High incidence of resorptions and fetal
lethality may be due to the direct action of radiation
on the fetuses or inhibitory action of radiation on the
protein synthesis and placental dysfunction (Abu
Gabal et al., 1994a,b; El Naggar et al., 1996).
The degree of damage induced by irradiation
depends on the degree of differentiation, state of the
cell concerning it’s cycle, the dose rate and the age of
the animal at the time of irradiation (Moustafa,
1997; Salama, 2004).
Morphological changes:
1- Uterine form and resorptions:
In the present study different abnormalities in
the uteri of pregnant female were noticed as unequal
distribution of embryos in the two horns,
implantation in one horn only, beside shrinkage of
the two horns and complete abortion in irradiated
pregnant rats on day 7 day of gestation. These results
are in line with those of Hong Wang et al. (1993) ;
Kafafy et al. (2006).
Ramadan (2007 b) found that pregnant
albino rats exposed to 3Gy (1Gy/3 times) on the 7th,
11th and 15th days of gestation showed severe
abnormalities, frequent implantation sites and
resorptions.
The present results obtained from pregnant
rats irradiated at 2Gy gamma rays on days 7 or 14 of
gestation and sacrificed on day 20 of gestation
resulted in numerous abnormal features. The
characters of variation are summarized as evidence of
abnormalities, implantation in one horn only, beside
unequal distribution of fetuses in the two horns,
presence of resorption leaving resorbed bodies
especially in the 14th day of gestation and reduction
in size of embryos. These results are supported by
other authors who reported more or less similar
results which varied with the variation of the dose or
the stage of development at which radiation was
performed (Prakash Hande and Uma-Devi, 1993;
El-Naggar et al., 1996; Ashry, 1997; Salama, 1998;
Kafafy et al., 2006., Ramadan, 2007a).
Bone marrow transplantation post-irradiation in
this study showed improvement in the uterine form
with equal distribution of embryos in the two horns in
the pregnant rats irradiated at 2Gy γ-rays on days 7 of
gestation, but in the pregnant rats irradiated at 2Gy γ-
rays on days 14 of gestation, unequal distribution of
Stem Cell 2012;3(3) http://www.sciencepub.net/stem
25
embryos in the two horns were noticed. These results
agree with those of Hussein (2004).
2- Embryonic malformations:
Teratogenitic effects observed in the present
work due to radiation exposure (R14and some cases in
R7 groups)were expressed as size diminution, growth
retardation, clubbed limbs, malformed eye lids,
malformed rostrum, and absence of some digits of
hands or toes of legs, twisted tail, short tail and
subcutaneous haemorrhage. These results agree with
those of Ramadan (2007b) who found that pregnant
albino rats irradiated with 0.5Gy 4 times on gestation
days 9,10,11,12, showed fetal interauterine death
together with serious teratogenic effects in the head,
eye and extremities of surviving fetuses and
Moustafa (2000) who reported that teratogenic
effects of γ-rays observed mainly in the fore and
hind limbs and tail region, ectodactyles and three
metacarpals.
The growth retardation recorded in the present
work agree with the results reported by many authors
(Hussein, 2004; Ramadan, 2007a ).
In the present study bone marrow
transplantation post-irradiation showed a slight
improvement in the size and length of fetuses on
pregnant rats irradiated at 2Gy γ-rays on days 7 or 14
of gestation. These results agree with those of
Hussein (2004).
2-Lung
A-Lung of the pregnant rats
In the present study exposure of the pregnant rats
to 2Gy of γ-rays on day 7 of gestation showed many
deleterious changes in lung tissue of the pregnant
rats, but, these changes were more pronounced on
day 14 of gestation. These changes include highly
thickened and congested alveolar septae, highly
elongated and branched bronchioles, their lumen
contained debris of degenerated epithelial cells with
ruptured epithelial lining of these bronchioles and
thick fibrous layers surrounding them, also highly
thickened, corrugated and distorted arterial walls
were observed with different masses of granuloma.
In this respect, Fleckenstein et al. (2010) stated
that chronic production of reactive oxygen and
nitrogen species is an underlying mechanism of
irradiation induced lung injury, they also noticed
increased lung damage and decreased immunity in
female rats post-irradiation (28Gy).
In 2005, Shediwah exposed male rats to 1Gy γ-
rays per day for 10 days and noticed many
histological changes in lung of these rats; these
changes include different masses of granuloma, fatty
degeneration, thickened and congested alveolar
septae, many fibrotic areas and distorted, elongated
and thickened walls of the bronchioles and the blood
vessels. Mahmod (2006) noticed that radiation
exposure caused many changes in the haemoglobin
such as decreased peptide bound and he noticed
many pathological changes in RBCs. He added that
increased mutation and decreased DNA content in
nuclei of the cells post-irradiation may be due to
production of active oxygen which lead to oxygen
pressure and increased free radicals which affect
chains of DNA that lead to cancer.
Abdollahi et al. (2005) noticed damaged
lungs of rats exposed to non-ionizing radiation
(20Gy). Through radiotherapy Petit et al. (2005)
noticed increased pneumonocytes 2-3 months in
females aged 50-60 years post radiation exposure. In
2005, Van der Meeren et al., demonstrated
increased immunological response in lungs of rats
exposed to 15Gy.
Lung damage and lung injury pneumonitis
post-irradiation was noticed by several authors (Liao
et al., 2000; El Khatib et al., 2009 ; Kirsch et al.,
2010; Miyake,2012).
Thickened and congested alveolar walls
congested and dilated blood vessels with haemolysis,
dilated bronchioles and appearance of different
masses of granuloma were realized in mice lung by
El Salkh (2009) post-exposure to EMF radiation.
Neutrophils are the first responding cells in lung
tissues injured by irradiation (Chianget al., 2005).
Lymphocytic alveolitis is a well-recognized
component that occurs in response to tissue injury
caused by irradiation, and aprominent feature of post-
radiation lung injury is the development of
lymphocytic alveolitis (Huang et al., 2002).
Dilated and congested blood vessels observed
in this study may be due to increased pulmonary
arterial pulse pressure noticed by Grant et al. (1988)
in rats exposed to radiation. Degenerated alveolar
septae and cells of the epithelial layer in the
bronchioles observed in the present study may be due
to highly affected DNA in the nuclei of their cells
(Simko, 2000 ; Zhang et al., 2006)
Bone marrow transplantation post-irradiation in
the present study showed a noticeable improvement
in the lung tissue of the pregnant rats especially on
day 7 of gestation, but highly thickened alveolar
septae and arterial walls were still observed on day
14 of gestation.
The present results revealed that in case of
injury, the stem cells from bone marrow are
responsible for tissue regeneration and these cells
have unique properties that make them attractive
candidates for the treatment of diseases and injuries.
Also stem cells can be transplanted to replace non-
functional or lost stem cells in tissues to accelerate
Stem Cell 2012;3(3) http://www.sciencepub.net/stem
26
tissue healing and restore the original function (Burt
et al., 2008).
In this study, highly increased collagen fibers
was detected in the lung tissue especially in the
thickened walls of the arteries, veins, bronchioles and
alveolar septae in the lung tissue of the pregnant rats
exposed to 2Gy γ-rays on day 7 or day 14 of
gestation.
Zhang et al .(2006) noticed many changes in the
lungs exposed to irradiation and they reported that
increased collagen post-radiation exposure may lead
to rapid healing, rapid differentiation of cells and
appearance of a new network of blood vessels.
According to Ahmadian et al. (2006) radiation
exposure of rats lead to increased collagen in the
skin. Khaki et al. (2006) reported that decreased
collagen, reticular fibers, ribosomes, glycogen
granules and cristae of mitochondria may led to
corrugated membranes.
Increased collagen post-irradiation exposure in
the different tissues was detected by several authors
(Shedwah, 2005; Al Gahtani, 2006; Eid and Al
Dossary, 2007; El-Salkh, 2009).
Rousovan et al. (1992) declared that the
increase in the collagen fibers may be due to
increased interstitial and white fibers under the effect
of radiation, but, Hassan et al. (1988) reported that
increased collagen fibers may lead to increase the
defense reaction against toxic materials.
The results of the present study showed nearly
normal collagen content in the lung tissue of the
pregnant rats treated with bone marrow post-
irradiation on day 7 of gestation, while, on day 14 of
gestation increased collagen fibers was detected in
the fibrotic areas and highly thickened alveolar
septae. Walls of blood vessels, bronchioles and areas
of granuloma cells showed increased affinity of
polysaccharides in the lung tissue of the pregnant rats
exposed to 2Gy of γ-rays on day 7 or day 14of
gestation with less stained debris of epithelial cells
inside the bronchioles and haemolysed RBCs.
Increased stain affinity of polysaccharides post-
irradiation in this work was also noted by many
authors (Shedwah, 2005; Al Dossary, 2007 ; El
Salkh, 2009). Increased stain affinity in granuloma
cells indicating the high content of polysaccharides in
these cells and increased stain affinity inside walls of
bronchioles, blood vessels, alveolar septae and
fibrotic areas may be due to increased thickness of
these components.
Decreased polysaccharides content in the
degenerated epithelial cells of bronchioles and
haemolysed RBCs was detected also by Abu El
Naga (1989) in lung tissue exposed to γ-rays.
Reduced glycogen in cells post-irradiation may be
due to decreased T3 and T4 hormones of the thyroid
glands, which lessen entrance of glucose to the cells
Results of the present study showed nearly
normal polysaccharides content in the lung tissue of
the pregnant rats treated with BM post-irradiation on
day 7of gestation, but, on day 14, thickened alveolar
septae, arterial walls and the fibrous layers encircling
the bronchioles acquired a dense stain affinity of
polysaccharides.
Concerning total protein, deeply stained walls
of arteries and brochioles, alveolar septae and
granuloma cells were observed in the lung tissue of
pregnant rats exposed to 2Gy of γ-rays on day 7 or
day 14 of gestation. This increase may be due to
increased thickness of the different walls, fibrotic
areas and increased areas of granuloma cells.
Increased total protein in lung tissue post exposure to
different types of radiations was noticed by many
authors (Gorczynsk and Wegrynowicz ,1991;
Shedwah, 2005; Al Dossary, 2007; El-Salkh, 2009;
Mansour, 2012).
Highly affected protein and DNA post-radiation
exposure may be due to response of hydrogen bounds
of these materials to radiation.
Bone marrow transplantation post-irradiation
in this study showed somewhat normal total protein
content in the lung tissue of pregnant rats on day 7or
day 14of gestation, but thickened a lveolar septae and
corrugated walls of bronchioles were deeply stained.
B- Lung of the embryos
In the present studyexposure of pregnant rats
to 2Gy of γ-rays on day 7 or 14 of gestation led to
many histopathological changes in the fetal lung
tissue. These changes were more drastic on day 14 of
gestation. These changes include: highly dilated and
congested blood vessels with numerous haemorregic
areas and common fibrosis around the walls of
bronchiole. The lumena of these bronchioles
contained debris of degenerated epithelial cells; the
congested alveolar septae lost their normal
architectures.
In agreement with the present results Abu El-
Naga (1989) studied the pathological changes in
embryos exposed maternally to 3Gy of γ-rays on the
6th and 10th days of the gestation. She noticed
thickened alveolar septae with reduced alveolar sacs
and highly dilated and ruptured walls of blood
vessels. She also noticed lots of haemrrhagic areas
and karyolysis in numerous nuclei.
Abu Gabal et al. (1998) observed pyknotic
nuclei in cells of the lung tissue of embryos
maternally exposed to 1Gy γ-rays, while those
maternally exposed to fractionated 2Gy showed some
delay in the development with thickening of their
inter alveolar septae.
Stem Cell 2012;3(3) http://www.sciencepub.net/stem
27
Also, Al-Dossary (2007) observed many
haemorrhagic areas covered the thickened alveolar
septae in the fetal tissue maternally exposed to EMF
radiation. She noticed many pyknotic nuclei in the
epithelial cells of the bronchioles with highly
thickened and corrugated arterial walls and
haemolysed blood cells inside them.
In the control fetal lung tissue, thin collagen
bundles are supporting the walls of the bronchioles,
blood vessels and alveolar septae, highly increased
collagen fibers was observed in the fetal lung tissue
maternally exposed to 2Gy γ-rays on day 7 or day 14
of gestation. Dilated walls of blood vessels, thickened
alveolar septae and walls of the bronchiole showed
dense stain affinity of collagen.
Results of the present study showed somewhat
normal distribution of collagen fibers was detected in
the fetal lung tissue maternally treated with 2Gy γ-
rays on day 7 or day 14 of gestation followed by bone
marrow treatment, but a slight increase of these fibers
was detected in the walls of the bronchioles, blood
vessels and alveolar septae on day 14 of gestation.
The regenerative potential of stem cells was
studied by several authors (Ferrari et al., 1998; Pye
and Watt, 2001; Kirsch et al., 2010).
The improvement observed in the fetal lung tissue
maternally exposed to γ-rays and treated with BM
may be due to the ability of bone marrow cells to
differentiate to mature, non-haematopoitic cells of
multiple tissues (Abedi et al., 2004).
Concerning polysaccharides, the fetal lung
tissue maternally exposed to 2Gy γ-rays on day 7 or
day 14 of gestation showed increased stain affinity.
Increased stain affinity of polysaccharides in
the fetal lung tissue exposed maternally to γ-rays may
be due to increased RBCs in the congested sinusoidal
spaces, blood vessels and haemorrhagic areas, since
RBCs contained 10% of their weight polysaccharides
(Junqueira and Carneiro, 2003). In accordance to
the present results Moustafa and Hafez (1998) and
Moustafa (2000) noticed an increase in PAS +ve
materials in the fetal tissues post exposure to 2Gy γ-
rays.
Eid et al. (1994) indicated that the frequency of
changes in polysaccharides content was high in lung
and ileum tissue of rat embryos exposed to 3Gy on
days 6 and 12 of pregnancy. Fetal lung tissue taken
from mothers exposed to 2Gy γ-rays on day 7 or day
14 of gestation followed by bone marrow
transplantation restorted the normal polysaccharides
content with a slight increase in stain affinity in walls
of the bronchioles, and alveolar septae of the fetal
lung tissue on day 14 of gestation.
In the present study increased stain affinity of
total protein content was observed in the fetal lung
tissue exposed maternally to 2Gy γ-rays on day 7 of
gestation. Faintly stained alveolar septae and densely
stained fibrous layers were detected on day 14 of
gestation. This increase in stain affinity of total
protein may be due to increased RBCs in the
congested alveolar septae and blood vessels or may
be due to appearance of the fibrous tissue, but
reduced stain affinity of total protein may be due to
damaged protein molecules by irradiation. This
finding is in accordance with those of Kapyaho et al.
(1983) who stated that ionizing radiation usually
inhibits the protein synthesis and the decline of
protein which they recorded could be attributed to the
degeneration in the cellular tissues.
In the present study somewhat normal total
protein content was detected in the fetal lung tissue
maternally treated with the bone marrow post-
irradiation on day 7 of gestation, while congested
arteries contained deeply stained RBCs. But on day
14 of gestation, highly thickened alveolar septae and
the fibrous layers surrounding the bronchioles
showed increased stain affinity of total protein.
It is clear that pregnant rats exposed on day 14
of gestation are more sensitive to γ-rays; BMT cannot
completely overcome radiation injury and restore the
normal content of collagen polysaccharides and total
protein content in the fetal and maternal lung tissue.
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... Ostrovsky et al. (2009) demonstrated oxidative stress and ROS stimulate cellular damage comprising apoptosis and DNA fragmentation. Dilated and congested blood vessels were realized in the present experiment and this may be due to increased pulse pressure of pulmonary arteries (Abuo El Naga & Abd Rabou, 2012). Also, the appearance of degenerated alveolar septae and debris of degenerated cells in the lung bronchioles may be related to of DNA effects in the nuclei of their cells (Zhang et al., 2006). ...
... Results of the current study revealed increased PAS +ve materials in the thickened walls of bronchioles, arteries and alveolar septae with moderately stained granuloma areas in lungs of pregnant rats of groups S1, S2 and revealed decreased PAS +ve materials in the fetuses of S1, S2 groups in a dose dependent manner as compared to control group. Such increasing PAS +ve materials inside walls of bronchioles, blood vessels, alveolar septae may be due to increased thickness of these components or may be due to the increase in the RBCs after toxicity as reported with Abuo El Naga & Abd Rabou (2012) and Abd El-Hady & Al Jalaud (2015). The decreased carbohydrate contents in the fetal groups of the current study may be due to the increased stress on the organs which leading to consuming high energy to equalize the pressure upon them. ...
... On the other hand, Abuo El Naga & Abd Rabou (2012) stated that the decreased protein content may be due to rupture of cellular organelles or to decreased ribosomes. Abdel-Meguid et al. (2012) stated that the decrease in protein content may be due to lysosomal membranes disruption under the effects of various toxicants which leads to releasing of their hydrolytic enzymes in the cytoplasm which causing dissolution and lysis of the target material within the cytoplasm. ...
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... One hundred and twenty voluntary male children (aging 6−12 years) have been chosen for the experimental work. They were divided into three groups as follows [1]: the first group served as control, the second group was exposed to electromagnetic field (EMF) emitted from the base station radiation, and the third group was exposed to electromagnetic field radiation and orally treated with 2.5 mL/day of olive oil during the experimental periods (5 weeks). The second and the third groups lived near the mobile phone base stations (100−150 m) more than 5 years and were exposed to electromagnetic radiation with constant power in the range of 1.4−4.7 mW/cm2, measured during the experiment. ...
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... Highly increased collagen fibres was detected in the liver and lung tissues of the pregnant rats and their fetuses exosed to 2Gy gamma rays on day 7 or day 14 of gestation. 61 Increased collagen post-radiation exposure in the different tissues was detected by several authors. 62,63,64 The present investigation is supported by the work done by those of Eid et al. 57 who revealed that newly born mice exposed to RFEMF from mobile phone 45min/day for one month showed increased collagen fibres in hepatocytes of the exposed group when compared to the control group. ...
... Reduced glycogen in cells post-irradiation may be due to decreased T3 and T4 hormones of the thyroid glands, which lessen entrance of glucose to the cells. 61 The reduction of PAS +ve materials was also noticed by Eid et al. 57 who observed asignificant decrease of of PAS +ve materials in the central and portal areas in liver of newly born mice exposed to RF-EMF from mobile phone (45min/day) for one month. ...
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... The accumulation and degeneration led to reduced protein content in the altered tissues [36] . Abu Elnaga and Abd Rabou [37] mentioned that decreased protein content due to broken cellular organoids or to reduced polyribosomes. The reduction in the protein content under the effect of many toxicants may be due to the disruption of lysosomal membranes which cause release of their hydrolytic enzymes in the cytoplasm and cause lysis and dissolution of the target material within the cytoplasm [38] . ...
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... In the present study, examination of fetal muscle fibres of groups S1 and S2 showed decreased density of total protein content. AbouEl Naga andAbdRabou (32) revealed that the decrease in protein content may be due to decreased ribosomes or rapture of cellular organelles. In addition, a decrease of protein content may be due to degenerated tissues and lysosomal membranes disruption by the effect of toxicants which leads to releasing of their hydrolytic enzymes in the cytoplasm which causing lysis of the target materials with it (33) . ...
... The accumulation and degeneration led to reduced protein content in the altered tissues [36] . Abu Elnaga and Abd Rabou [37] mentioned that decreased protein content due to broken cellular organoids or to reduced polyribosomes. The reduction in the protein content under the effect of many toxicants may be due to the disruption of lysosomal membranes which cause release of their hydrolytic enzymes in the cytoplasm and cause lysis and dissolution of the target material within the cytoplasm [38] . ...
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