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J Matern Fetal Neonatal Med, 2013; 26(11): 1143–1146

! 2013 Informa UK Ltd. DOI: 10.3109/14767058.2013.770463

ORIGINAL ARTICLE

Neuronal apoptosis in the neonates born to preeclamptic mothers

Hese Cosar1, Erdener Ozer2, Hande Topel3, Zelal Kahramaner1, Ebru Turkoglu1, Aydin Erdemir1, Sumer Sutcuoglu1,
Alper Bagriyanik3, and Esra Arun Ozer1
1
Tepecik Training and Research Hospital, Neonatology Clinic, Yenisehir, Izmir, Turkey, 2Department of Pathology, Dokuz Eylul University School of
Medicine, Izmir, Turkey, and 3Department of Histology and Embryology, Dokuz Eylul University School of Medicine, Izmir, Turkey
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Abstract Keywords
Objective: Preeclampsia may result in uteroplacental insufficiency and chronic intrauterine fetal Animal model, apoptosis, newborn, perinatal
distress. The aim of this study is to address this issue investigating neuronal apoptosis in an asphyxia, preeclampsia
experimental model of preeclampsia and to evaluate the neurological outcome of the perinatal
asphyxia in the neonates born to preeclamptic mother. History
Materials and methods: Two out of four pregnant Sprague–Dawley rats (preeclamptic group)
were given water containing 1.8% NaCl on gestation day 15 and 22 in order to establish the Received 27 December 2012
model of preeclampsia whereas other two (non-preeclamptic group) received normal diet. Accepted 23 January 2013
A model of perinatal asphyxia was established on the postnatal 7th day to one preeclamptic Published online 6 March 2013
and one non-preeclamptic dam. Overall 23 pups born to overall four dams were decapitated
to assess neuronal apoptosis by the TUNEL assay.
For personal use only.

Results: The number of apoptotic neuronal cells was significantly higher in the preeclampsia
groups in comparison with the control group (p ¼ 0.006 and p ¼ 0.006, respectively). It was also
significantly higher in the asphyctic/non-preeclamptic group than the count in the control
group (p ¼ 0.01). There was also significant difference between both asphyctic groups
(p ¼ 0.003).
Conclusion: We conclude that preeclampsia causes small babies for the gestational age and
cerebral hypoplasia. Both preeclampsia and perinatal asphyxia can cause increased neuronal
apoptosis in the neonatal brains. However, the prognosis for neurological outcome is much
worse when the perinatal asphyxia occurs in newborns born to preeclamptic mothers.

Introduction can result in neuronal apoptosis in the newborn brain and to

Preeclampsia is a multisystemic clinical condition character-
ized by hypertension, proteinuria and edema in pregnant
women. It is one of the major causes of fetometarnal
morbidity and mortality and affects 2%–8% of all pregnancies
[1,2]. It can lead to severe fetal complications, such as fetal
growth retardation, premature birth and placental abruption
due to uteroplacental insufficiency [3,4].
Perinatal asphyxia is a common problem seen in the
newborn period. Hypoxia and ischemia cause primary
neuronal damage by neuronal necrosis and infarction during
perinatal asphyxia [5,6]. It has also been reported that
perinatal asphyxia can induce a cascade of intracellular events
leading to cell apoptosis by activation of proapoptotic genes
and caspase-3 [7].
Whether preeclampsia can cause the neuronal damage and
apoptosis in the perinatal period by systemic hypoperfusion
and ischemia is still not clear. Therefore in this study, we
aimed to investigate in an animal model whether preeclampsia
Address for correspondence: Dr Esra Arun Ozer, Associate Professor of
Pediatrics and Neonatologist, Tepecik Training and Research Hospital,
Neonatology Clinic, Yenisehir, Izmir, Turkey. Tel: + 90 232 4696969
(3509). Fax: + 90 232 4330756. E-mail: eozer@deu.edu.tr
evaluate the neurological outcome of the perinatal asphyxia in
the neonates born to preeclamptic mother.
Materials and methods
Experimental procedure
The present study was approved by the Aegean University 13
20
Local Ethical Committee on Animal Research. Overall
four pregnant Sprague–Dawley rats were included in the
study. The animals were kept at controlled temperature
(21  C–23  C) and 12 h light–12 h dark condition. They had
free access to food and tap water until day 15 of pregnancy
when the drinking water was modified for the experimental
model. The animals were mated. Day 0 of pregnancy was
defined when the vaginal plaque was seen. Two dams were
selected as the control group and received a normal diet.
The other two were included in the preeclamptic group
and received water containing 1.8% NaCl between gestation
day 15 and 22 after fertilization for the development of
preeclampsia model [8]. On postnatal day 7 overall 23 pups
were separated into four groups:
Group 1: The pups born to the dams with preeclampsia and
exposed to asphyxia on postnatal day 7 (n ¼ 6).
 1144 H. Cosar et al. J Matern Fetal Neonatal Med, 2013; 26(11): 1143–1146

Group 2: The pups born to the dams with preeclampsia and Table 1. The comparison of the groups in terms of gender, body and
brain weights.
were not exposed to asphyxia (n ¼ 5).
Group 3: The pups born to the dams without preeclampsia Preeclamptic Non-preeclamptic
and exposed to asphyxia on postnatal day 7 (n ¼ 6). group (n ¼ 11) group (n ¼ 12) p
Group 4 (control): The pups born to the dams without
Gender (Female/Male) 6/5 6/6 0.82
preeclampsia and were not exposed to asphyxia (n ¼ 6). Body weight (g)* 8.1  1.2 10.6  0.6 50.001
Each group was randomly assigned and female–male Brain weight (g)* 0.45  0.03 0.56  0.04 50.001
difference was not considered. Body weight and sex of the
*Values are presented as means  SD.
pups were recorded. For the experimental model of perinatal
asphyxia on postnatal day 7, the pups in group 1 and 3
were anesthetized by intraperitoneal ketamine (50 mg/kg) þ Table 2. Neuronal apoptotic cell count by the
TUNEL assay.
xylazine (10 mg/kg). A median incision was made in the neck.
The left carotid artery was identified after dissection and was Apoptotic cell count*
ligated with a 5/0 silk suture. After a 1–2 h recovery period
animals were placed in transparent fiberglass chamber and Group 1 (n ¼ 6) 12.6  5.89
Group 2 (n ¼ 5) 8.4  2.60
exposed to a continuous flow of 8% oxygen and 92% Group 3 (n ¼ 6) 6.3  2.16
nitrogen for 2.5 h to induce systemic hypoxia. Following a Group 4 (n ¼ 6) 2.5  2.07
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6 h reoxygenation period in the room air, under high-dose

ketamine (500 mg/kg) anesthesia all pups were decapitated.
The brain tissues were obtained for the pathological evalu- Table 3. Statistical comparison of groups accord-
ing to the neuronal apoptotic cell count.
ation and brain weights were recorded.
Groups p
Histopathological evaluation
Group 1 versus Group 2 0.24
Brain tissues were fixed in 10% buffered formalin for 3 d. Group 1 versus Group 3 0.03
The brains were sectioned coronally through the hippocampus Group 1 versus Group 4 0.006
according to the rat atlas [9]. Following the embedding in Group 2 versus Group 3 0.23
Group 2 versus Group 4 0.006
paraffin, the tissue blocks were cut into a series of 5 m
For personal use only.

Group 3 versus Group 4 0.01

thickness at poly-L-lysin-coated slides for the application of
Apop TagÕ in situ apoptosis detection kit S7101 (Millipore,
Billerica, MA). The tissue sections were deparaffinized and
pretreated with proteinase K (20 mg/mL). Endogen peroxidase
activity was blocked using 3.0% hydrogen peroxide in
Phosphate Buffered Solution (PBS). Equilibration buffer
(75 mL/5cm2), working strength TdT enzyme (55 mL/5cm2),
stop/wash buffer and anti-digoxigenin conjugate were applied,
respectively. Peroxidase substrate was applied subsequently to
develop color. The sections were counterstained in 0.5% (w:v)
methyl green and mounted before viewing under microscope.
Apoptotic neuronal cells in the samples were labeled
(brown color) and counted in the region of the hippocampus
CA1, CA and CA3 of both right and left hemispheres by two
investigators (E.Ö. and H.T.) in a double-blinded manner. The
‘‘hot spot’’ areas at 10 high power fields were selected under
the light microscope for the apoptotic cell count. The mean
number of the count was recorded for each sample.
Statistical analysis
Results were expressed as means  SD. One-way ANOVA
and Mann–Whitney U tests were used for multiple compari-
sons. p50.05 was considered as statistically significant.
Interobserver correlation was assessed according to the kappa
values as described in the literature [10], such as 0.01–0.20:
no correlation; 0.21–0.40: low correlation; 0.41–0.60: mod-
erate correlation; 0.61–0.80: high correlation; 0.81–1.00: very
high correlation.
Results
The mean body and brain weights of the preeclamptic dams’
pups were found significantly lower than those of
non-preeclamptic group (p50.001). There was no statistical
difference between the groups in terms of gender (Table 1).
Interobservation correlation for the count of apoptotic
neuronal cells was high between investigators (Kappa value:
0.74). The mean  SD of apoptotic cells in all four groups are
demonstrated in Table 2.
The count of neuronal apoptotic cells was statistically
demonstrated in Table 3 and illustrated in Figure 1. The
apoptotic cell count was significantly higher in Group 1
(preeclampsia-asphyxia) and Group 2 (only preeclampsia)
compared to the count in Group 4 (control) (p ¼ 0.006
and p ¼ 0.006, respectively). The count in Group 1 was
also significantly higher in comparison with Group 3 (only
asphyxia) (p ¼ 0.03) and in Group 3 compared to Group 4
(p ¼ 0.01). No statistically significant difference was found
between Group 2 and both Group 1 and 3.
Discussion
We aimed in this study to investigate neuronal apoptosis
in an experimental model of preeclampsia and to evaluate
the neurological outcome of the perinatal asphyxia in the
neonates born to preeclamptic mother.
Preeclampsia characterized by decreased uteroplacental
blood flow and ischemia is an important risk factor in the
development of intrauterine growth retardation (IUGR) and is
the most common cause of IUGR without anomalies. Fetal
growth retardation is seen in 28% of pregnancies complicated
with hypertension and preeclampsia [11]. Our results in the
present study were in consistent with this observation.
 DOI: 10.3109/14767058.2013.770463
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Figure 1. Neuronal apoptotic cells labeled by the TUNEL assay. Note the higher counts in both preeclamptic groups.
asphyxia, PE: Preeclampsia.
For personal use only.
We found that the body weights of neonates born to the
preeclamptic mothers were significantly lower. Therefore, in
accordance with the relevant clinical studies, preeclampsia
can cause IUGR by affecting fetal growth adversely.
Neurodevelopmental disorders have been found more often
in clinical trials for the infants exposed to preeclampsia. In a
study including 2303 newborn, the risk of cerebral palsy was
found 1.5 times higher in these infants [12]. In another study
comparing the neurological outcomes between premature
newborns in terms of preeclampsia and premature rupture of
membranes, poor neurological outcomes were found higher in
the infants born to preeclamptic mothers [13]. The authors
have concluded that prolonged exposure to the intrauterine
distress due to placental insufficiency in preeclampsia
increases the incidence of poor neurologic outcomes in the
newborns. Similar to the data from the clinical studies,
we observed that preeclampsia increased neuronal damage
by causing apoptosis in the pups born to preeclamptic dams.
Perinatal asphyxia is the most common cause of neuro-
logical sequelae in the perinatal period and one of the major
perinatal problems of the newborns. Perinatal asphyxia causes
primary neuronal damage, including cell necrosis and infarc-
tion by hypoxia and ischemia [5,14]. The increase of free
oxygen radicals and the intracellular Caþ2, and activation of
apoptotic pathways are the major events occurring in the
second phase of the neuronal damage.
Insufficient interstitial cytotrophoblast invasion and super-
ficial endovascular invasion in preeclampsia result in placen-
tal hypoperfusion and ischemia. Oxidative stress, increase
in reactive oxygen radicals and deficiency of antioxidant
defense are shown as a cause of neuronal injury in
preeclampsia [15–17]. Although there are a few data
suggesting the apoptotic effect of preeclampsia in the placenta
Preeclampsia and perinatal neuronal injury 1145
C: Control, PA: Perinatal
and fetus, such as increased trophoblast apoptosis [18] and
increased fetal DNA in the plasma of pregnant women with
preeclampsia [19,20], the data are not convincing yet to
suggest that the newborns born to preeclamptic mothers are
prone to cerebral neuronal apoptosis. But the results of our
study clearly show that the count of neuronal apoptotic cells is
significantly higher in the preeclamptic group. We therefore
conclude that preeclampsia causes neuronal apoptosis and
also worse neurological outcome due to the oxidative stress
and ischemic injury resulting from the impaired placental
circulation in the intrauterine period occurring in
preeclampsia.
Prematurity and intrauterine growth retardation are the
important problems that can affect the prognosis of infants
born to preeclamptic mothers [21,22]. Increased cerebral
neuronal apoptosis was shown in an experimental model of
intrauterine growth retardation and the neuronal damage
became worse when perinatal asphyxia was applied addition-
ally to the model [23]. Similarly, our study showed that
although apoptotic neuronal cell count was higher in the
perinatal asphyxia model, the increase in the neuronal
apoptosis appeared much evident when it was combined
with the preeclampsia model.
In conclusion, the present study is the first experimental
study that investigates the cooperative effect of perinatal
asphyxia with preeclampsia on neuronal apoptosis, in other
words neuronal damage. We show that preeclampsia causes
small babies for the gestational age and cerebral hypoplasia.
In addition both preeclampsia and perinatal asphyxia can
cause increased neuronal apoptosis in the perinatal period.
However, the prognosis for neurological outcome is much
worse when the perinatal asphyxia occurs in the newborns
born to preeclamptic mothers. Further studies may focus on
 1146 H. Cosar et al.
the preventive strategies to protect infants born to preeclamp-
tic mothers from perinatal asphyxia and protocols to follow up
them for the neurodevelopmental status.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
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