How to cite this article: Hegazy A A, Ibrahim H I, Sabry R M, Abass E S. Effect of Gestational Exposure to Monosodium Glutamate on the Structure of
Fetal Rat Lung. Anatomy Physiol Biochem Int J. 2017; 3(2): 555609. DOI:10.19080/APBIJ.2017.03.555609.
The aim of this work was to elucidate the possible developmental changes in the offspring lungs induced by administration of monosodium glutamate (MSG) to the pregnant albino rats. Forty pregnant albino rats were used in this study, equally divided into two groups. The first group was the control one, administered distilled water by gastric tube; the second was instead given MSG dissolved in distilled water. The animals were sacrificed at different four days of gestations; 14th (GD14), 16th (GD16), 19th (GD19) and 21st (GD21). Fetal lung sections were stained with Haematoxyline and Eosin (H&E) and with proliferating cell nuclear antigen (PCNA) reactions. The obtained data were statistically analyzed. At GD14, the bronchial tubes started to branch to form small simple primitive bronchiolar tubules. They were lined with pseudo stratified epithelium. At GD16, the bronchial tubes were lined with columnar epithelium with cytoplasmic vacuolization giving a glandular appearance. At GD19, the lining of tubular air spaces was cuboidal epithelium. GD21 showed the formation of secondary crests and appearance of air saccules lined with flat epithelium. Treated animals showed the epithelium of these tubules with desquamation and necrotic cells. Also, there was marked growth retardation in comparison to the control one. The PCNA immune histochemical staining showed high positive immunoreactions in the bronchiolar epithelium and the adjacent stroma. This is more evident in the treated subgroups than in the control ones. It has been concluded that administration of MSG to mothers during pregnancy may exert some growth retardation and histopathological changes in the fetal lungs.
Keywords: Lung Development, Rat, Monosodium glutamate, PCNA.
Lung diseases represent one of the leading causes of worldwide morbidity and mortality. The problem is expected to be worse in the future . This necessitates investigating the possible environmental factors, habits and food types that might be engaged in pathogenesis of pulmonary diseases, particularly during the fetal life. This of particular importance as many drugs and substances taken by (or given to) the pregnant mothers can cross the placental barrier to affect their fetuses . One of such substances is monosodium glutamate (MSG) that might affect the lungs of fetal mice after exposure of their mothers to it . Various developmental changes in the animals’ offspring tissues might occur following MSG treatment . Moreover, the administration of MSG in high concentrations for long period of time triggers the production of reactive oxygen species (ROS) coupled with impaired oxidant/ antioxidant balance leading to a state of oxidative stress . The lung is more susceptible to oxidative injury than any other organ in the body because of constant exposure to air that might contain toxic particles or oxidant gases such as nitrogen oxide or ozone .
MSG is a well-known flavor enhancer used by both Western and Eastern populations. It is responsible for the delicious taste; and it is incorporated into a large number of solid and liquid foods. Experimental studies have established that the added MSG in foods influences palatability and preference . Nowadays, MSG is commonly sold in the Egyptian supermarkets. Many Egyptians consume MSG in both ready and home-made foods, almost daily .
MSG contains glutamic acid (78%), sodium (22%) and water . Glutamate receptors are present in the central nervous system as the major mediators of excitatory neurotransmission. Neural injury associated with trauma, stroke, epilepsy and many neurodegenerative diseases might be mediated by excessive activation of such receptors. Several subtypes of glutamate receptors are demonstrated in the rat and monkey heart, with preferential distribution within the conducting system. The receptors are also present in the kidney, liver, lung, spleen and testis . Juvenile obesity, reduced general activity and learning disability have been reported in rat and mouse offspring that received MSG through their mothers during pregnancy . Moreover glutamate in high doses produces neuroendocrine
abnormalities and neuronal degeneration . It also might be
engaged in oxidative damage of many organs such as rat thymus
and kidney [13-15].
Many questions have been raised about the role of MSG in
provocation of asthma. It has been observed that MSG induces
signs and symptoms of asthma . Previous studies reported
cases of asthma following MSG ingestion through food. The
reaction to MSG was noticed to be a dose dependent; and it
might be delayed up to 12 hours, making its recognition difficult
for both patient and physician [17,18]. PCNA is a 36 kd protein
involved in protein synthesis; and can be considered as an early
indication of deviations to normal functioning .
The aim of this work was to elucidate the changes that might
take place in the developing lung after oral administration of
MSG to the pregnant mothers of the albino rats using routine
histological and PCNA immunohistochemical investigations.
This study was carried out on forty adult female and twenty
adult male albino rats of 200-250 gm body weight (BW). They
were reared in the animal house, Faculty of Medicine, Zagazig
University. The experiment was performed according to the
norms of the ethical committee of the University.
Adult females were caged with adult males for mating, at
ratio of 2:1 respectively in the cage. Vaginal smear was done for
each female rat at the morning. The day in which the smear was
sperm positive was considered as the zero day of pregnancy. The
females with negative vaginal smear were isolated and re-kept
again with males. The process was repeated until all females
Pregnant females were equally divided into two groups;
each contains 20 animals. The first group was the control one,
administered distilled water by gastric tube; the second was
instead given MSG in a dose of 2mg/kg BW, dissolved in distilled
water. The animals were sacrificed using ether inhalation, at
different four days of gestations; 14th (GD14), 16th (GD16), 19th
(GD19) and 21 (GD21).
Their embryos were extracted; and lung tissue specimens
were retrieved and processed for light microscopic examination
. Sections were stained with H&E for study of the histological
changes; and with PCNA for immunohistochemical examination.
The data were obtained using Leica Qwin 500 image analyzer
computer system (England) at the Histology Department, Qasr
Eleini Faculty of Medicine. The image analyzer consisted of
colored camera, monitor and hard disc of IBM personal computer connected to the microscope, and controlled by Leica Qwin 500
software. The image analyzer was first calibrated automatically
to convert the measurement units (pixels) produced by the
image analyzer program into actual micrometer units.
The optical density of immunoreactions of PCNA positive
cells was measured in intersaccular and interstitial cells.
The optical density was expressed in the form of maximum,
minimum grey as well as sum of grey and mean grey parameters.
Data obtained about the optical density of PCNA in the epithelial
and interstitial cells for the control and treated groups were
analyzed statistically using ANOVA Test. The statistical analysis
was done using SPSS software (version 19.0; SPSS, Chicago, IL).
The p ≤ 0.05 was considered to indicate a statistically significant
Light microscopic examination of the rat lung of control
group at GD14 showed the bronchial tubes start to branch
forming simple primitive bronchiolar tubules. They were
lined with pseudostratified epithelium (Figure 1). The
PCNA immunohistochemical staining showed high positive
immunoreactions in the bronchiolar epithelium and the adjacent
stroma (Figure 2). The rat lung of control group at GD16 showed
that the bronchial tubes were lined with columnar epithelium
with cytoplasmic vacuolization giving them the glandular
appearance (Figure 3). The PCNA immunohistochemical staining
showed high positive immunoreactions in the bronchiolar
epithelium and the adjacent stroma (Figure 4).
The rat lung of control group at GD19 showed the appearance
of the tubular air spaces lined with cuboidal epithelium (Figure
5). The PCNA immunohistochemical staining showed moderate
positive immunoreactions in the bronchiolar epithelium and the
adjacent stroma (Figure 6).
At GD21, the lung of control group showed the formation
of secondary crests and appearance of air saccules. They were
separated by intersaccular septa and lined with flat epithelium
(Figure 7). The PCNA immunohistochemical staining showed
mild positive immunoreactions in the bronchiolar epithelium
and the adjacent stroma (Figure 8).
At GD14, the lung of treated group showed simple primitive
bronchiolar tubules; and the epithelium of these tubules
showed desquamation and necrotic cells (Figure 9). The
PCNA immunohistochemical staining showed high positive
immunoreactions in the bronchiolar epithelium and the adjacent
stroma (Figure 10).
The rat lung of treated group at GD16 showed bronchiolar
tubules lined with pseudostratified epithelium with abnormal
cells showing marked disintegration of their nuclei. There were
necrotic cells. There was discontinuation in different parts of the
epithelium; and picnotic dark stained nuclei were seen (Figure
11). The PCNA immunohistochemical staining showed high
positive immunoreactions (higher than the previous group) in
the bronchiolar epithelium and the adjacent stroma (Figure 12).
The lung of treated group at GD19 showed marked growth
retardation with delayed development of the lung in comparison
with the control one. Only bronchial tubes and small number
of the primitive bronchiolar tubules were developed and lined
with pseudostratified epithelium with marked desquamation to
their epithelium (Figure 13). The PCNA immunohistochemical staining showed high positive immunoreactions (higher than the
previous group) in the bronchiolar epithelium and the adjacent
stroma (Figure 14).
The lung of treated group at GD21 showed marked congestion
and dilatation of the blood vessels and areas of hemorrhage were
seen. There was marked thickening of the intersaccular septa
and narrowing of most of the saccules. These saccules were lined
with cuboidal epithelium not flat as the control group of the
same age and no secondary crests and no capillaries were seen
in comparison with the control group of the same age (Figure
15). The PCNA immunohistochemical staining showed intense
high positive immunoreactions (higher than the previous group)
in the bronchiolar epithelium and the adjacent stroma (Figure
There was marked increase in the PCNA positivity in all
treated subgroups with the increase at the rat embryo age. It
reached to the highest positivity at GD21. The percentage of
tissues stained per 400 high power field in PCNA stained slides
between the control and treated subgroups showed highly statistical significant differences at the GD19 and the GD21 (p
<0.001) and there was statistically significant difference at the
GD16 (p <0.01), but that there was no statistical difference
between the control and treated subgroups at the GD14 in area
% of lung tissue (p =0.11) (Table 1); (Figure 17).
In this work, albino rat was used as an experimental model
as it is easy to be bred, operated and injected. Moreover, the
animal model makes it possible to follow the changes from initial
diagnosis to the first sign of organ impairment .
In the present study, the fetal rat lung treated with MSG at
GD14 showed a decrease in the branching of the bronchial tubes
into simple primitive bronchiolar tubules in comparison with the
control group of the same age; and they were also situated within
abundant loose mesenchyme. The bronchial tube was lined with
pseudostratified epithelium, but some cells showed cytoplasmic
vacuolation. Also, there were some cells desquamated inside the
bronchial lumen forming cellular debris. These findings are in
agreement with Lieberthal and Levine  who attributed the
vacuolation of the cytoplasm to the cellular necrosis that causes
impaired membrane transport activity and results in influx of
sodium and water into the cell. This results in cell swelling,
a characteristic early marker of necrosis, as well as loss of
functional and structural integrity of the plasma membrane.
In the present study, the histological examination of the fetal
rat lung treated with MSG at GD16 showed in comparison with
the control group of the same age a decrease in the progression
of the bronchial tubes into primitive bronchiolar tubules. The
bronchial tube was lined with pseudostratified epithelium in
some parts, but there were some epithelial cells contained
deeply stained nuclei with no cytoplasmic vacuolation which
is characteristic to this stage of development as in the control
group. There were some epithelial cells that were markedly
desquamated inside the bronchial lumen. This delay in the
lung development could be a result of MSG given to the rat
mothers during pregnancy. The cause of growth suppression
after MSG treatment is thought to be brought about by the
impaired production of growth hormone releasing factor, which
accompanies the necrosis of nerve cells in the arcuate nucleus
Another bronchial tube showed that its epithelium was
consisted of abnormal cells showing marked disintegration of
their nuclei (necrotic cells) and picnotic dark stained nuclei
were seen (a marker for apoptosis). Pavlovic et al.  said that
one of the mechanisms involved in MSG-induced apoptosis was
the down-regulation of Bcl-2 protein expression. MSG-induced
apoptosis and altered level of Bcl-2 protein are also related with
oxidative stress. Namely, the treatment of animals with MSG
resulted in an increase in the oxidative stress within the kidneys,
liver, brain and lung; and this presented a possible mechanism
of cell toxicity.
The fetal rat lung treated with MSG at GD19 showed a
retardation of development of the lung in comparison with
the control one. Only bronchial tubes and small number of the
primitive bronchiolar tubules were developed among abundant
loose mesenchymal tissue. No branching of the bronchiolar
tubules occurred and no tubular air spaces were developed
as in the control group of the same age. This delay in the lung
development is in general agreement with Millard et al. .
The bronchial tube was lined with pseudostratified epithelium
and picnotic dark stained nuclei were seen (apoptosis). Some
cells of the epithelium were disrupted and desquamated inside
the lumen forming cellular debris. This is in agreement with
Pavlovic et al. .
The primitive bronchiole in this group was lined with
pseudostratified epithelium not cuboidal epithelium as the
control group of the same age; and there were necrotic cells
showing disintegration of their nuclei in the lining epithelium and
discontinuation of the epithelium was seen. So, the lung of this
group was still in the pseudoglandular stage not the canalicular
stage as the control group of the same age. Also, Hildeman et al.
. Mentioned that the excessive generation of reactive oxygen
species (ROS) in the cells is known to damage DNA, lipids and proteins. Lipid peroxidation in cellular membranes damages
the polyunsaturated fatty acids especially in lymphoid cells, and
sensitizes T cells to apoptosis by decreasing the expression of
In the present study, the histological examination by
H&E of the fetal rat lung treated with MSG at GD21 showed
marked congestion and dilatation of the blood vessels and they
were lined with a layer of flat endothelial cells. Many areas of
hemorrhage were also present. Oda et al.  (said that this
hemorrhage indicates separation of endothelial cell junction.
However, Martin said that this hemorrhage is due to increase in
the number of neutrophils in the blood capillaries that damage
the epithelium by secreting many injurious substances such
as oxygen derived free radicals and lysosomal enzymes .
Also, there was marked edema around the congested blood
vessels and in the interstitium. Doohan  reported that the
thickening and expansion of the interstitium was due to the
interstitial edema resulting from histamine release from the
immune cells which led to increase the infiltration rate and
protein leak out from the blood capillaries into the interstitium.
Also, there was marked thickening of the intersaccular septa and
marked narrowing and collapse of most of the saccules. This
thickening in the intersaccular septa was due to marked cellular
infiltration. Other authors reported similar findings; and added
that the inflammatory cellular infiltration was related to the
oxidative stress resulting in generation of mediators such as IL-8
and cytokine-induced neutrophil chemoattractant that attract
the inflammatory cells into the pulmonary microcirculation and
then to the air spaces due to destruction of the endothelial cells
and the capillary membrane by the free radicals .
Some of these collapsed saccules were lined with cuboidal
epithelium only with no transition to flat epithelium as in the
control group of the same age and there was discontinuity of the
epithelial lining of some of these saccules, also the bronchioles
were lined with epithelial cells showing marked disintegration
of their nuclei (necrotic cells) and there was marked disruption
and discontinuity of its epithelium with cytoplasmic vacuolation.
The increase of lipid peroxidation following MSG treatment is an
important factor causing endothelial cell damage as the increase
in lipid peroxidation leads to loss of membrane integrity and cell
degeneration . On the other hand, another study attributed
the endothelial cell damage to the direct effect of the circulating
toxins or to the release of vasoactive cytokines from macrophages
and platelets or from the release of lysosomal enzymes .
Also, these results are in agreement with others who stated
the hypertrophy of the epithelial cells lining the bronchioles which
might be distended by mucus . The increase in mucus and
the hypertrophy of mucus secreting cells represent an important
cause of airflow obstruction, decreased alveolar ventilation or
hypoxia and consequently asthma. This observation might
explain the state of asthma which was reported by many authors
following MSG treatment.
In the present study, the immunohistochemical staining
using PCNA stain showed that in the control group at both
GD14 and GD16, there was high positive immunoreactions for
PCNA in the bronchiolar epithelium and the adjacent stoma,
but at GD19 there was moderate positive immunoreactions for
PCNA in the bronchiolar epithelium and the adjacent stoma
and finally at GD21 there was mild positive immunoreactions
for PCNA in the bronchiolar epithelium and the adjacent stoma
as the cell proliferation of the lung is increased during the
peudoglandular stage and begin to decrease in the canalicular
stage then markedly decreased during the saccular stage also
found that total cell proliferation of fetal rat lung declined during
gestation . They added that there was a relative decrease
of epithelial proliferation during the latter stages of gestation,
whereas endothelial cell proliferation increased during this
time. Differentiation of type II alveolar epithelial cells to type I
alveolar epithelial cells was associated with decreased epithelial
In the group treated with MSG, there were high positive
immunoreactions for PCNA at all age subgroups in comparison
with the control ones of the same ages. This marked increase
in PCNA reactions in all treated group might be due to the
marked cell degeneration, damage and delayed maturation that
occurred in these groups, so these cells try to proliferate more
to compensate this degeneration and damage. Similar findings
were reported by who said that increased cell proliferation,
particularly alveolar type II epithelial cells, is characteristic of
several acute and chronic pulmonary injuries . The results
of the present study were confirmed by statistical analysis
of the morphometric study. The percentage of PCNA stained
tissue/unit area showed that there was marked increase in
all treated groups compared with the control one of the same
ages. This could be an indication of delayed maturation and/or
Administration of MSG during pregnancy and the period
of organogenesis might exert histopathological changes in the
fetal lungs. Therefore, it might be suggested to minimize or
even prohibit eating foods containing MSG especially during
pregnancy to avoid the occurrence of such possible hazards.
Future studies are recommended using larger numbers of
animals with different doses to define at which one the hazard
effect could be inevitable.