Faculty of Medicine & Health Sciences

Neural Tube Defects (NTD): A Mouse Model of Hyperthermia-Induced

Exencephaly
Padmanabhan R, Department of Anatomy, E -mail:padmanabhanr@uaeu.ac.ae
Shafiullah M, Department of Pharmacology
Tariq S, Electron Microscopic Unit
U.A.E. University, Al-Ain, P.O. Box: 17666, U.A.E.

Abstract
The etiology of NTD remains largely unknown. Among the environmental agents causally implicated in
NTD, hyperthermia has the dubious distinction of first being discovered as a neuroteratogen in laboratory
animals and then in hum ans. However, the morphologic basis of hyperthermia-induced NTD is poorly
understood. The objective of our study was to establish an animal model in which a high frequency of NTD
could be induced in order to investigate the pathogenetic mechanism. Our earlier experiments in rats
showed that maternal hyperthermia at critical stages of neural tube formation resulted in a low frequency of
NTD. In the present study, we exposed pregnant TO mice for 9 minutes on gestation day 8 to an elevated
temperature of 43°C in a water bath by immersing them up to their neck. The controls were sham -treated at
37.5°C. The embryos were scored for exencephaly and axial skeletal abnormalities on GD 18. A total of 57
litters were examined. Mice sham treatment both once and twice on GD 8 had a low incidence of embryo
resorption (2-3%) and 0%incidence of intrauterine growth retardation (IUGR). There were no fetal
abnormalities. In contrast hyperthermia induced a significant reduction in mean fetal body weight, a dose-
dependent increase in resorption (21-28%) and a 90-100% incidence of IUGR at -2SD level..
Hyperthermia-induced exencephaly affected 16% and 46% of live fetuses of the single and double dose
groups respectively. These malformations were accompanied by several axial skeletal abnormalities.
These data indicate that TO mouse is a convenient model for further investigation into the pathogenetic
mechanisms of exencephaly.

1. INTRODUCTION
Dareste [1] was possibly the first investigator to demonstrate the deleterious effects of hyperthermia on the
developing embryos. It has now been well established that maternal heat stress during gestation can
produce a variety of fetal malformations in humans as well as in laboratory animals [2-8]. The spectrum of
anomalies has been observed to depend on the species, strain, developmental stage and duration and degree
of heat exposure. The developing central nervous system appears to be particularly susceptible to the
teratogenic effects of maternal hyperthermia [4, 5, 6, and 7]. Bot h gross anomalies such as anencephaly,
encephalocele and subtle histological abnormalities have been reported to occur. Sensitive periods for
various heat-induced anomalies have been reported [9-10]. Hyperthermia has been reported to induce
several isoforms of heat shock proteins [11, 12], and augment apoptosis in selected embryonic primordia of
organs [13]. Homeotic transformations have been observed in the developing axial skeletal structures of
heat -treated rat embryos [14]. In our recent attempt to study pathogenetic mechanisms of hyperthermia-
induced neural tube defects and axial skeletal abnormalities, we used TO strain of mouse. To the best of
our knowledge, this strain has not been used in any other reproductive toxicology laboratories. Although
our previous studies have discovered that TO strain has a particular susceptibility to a variety of teratogens
such as ethanol, aspirin, valproic acid and retinoic acid [15-19], it was not responding to hyperthermia in
terms of NTD when heat was applied for 1 hour at 43°C [unpublished data, Padmanabhan and Shafiullah].
The objectives this work was to attempt a variety of strategies to test the sensitivity of embryos of TO strain
to maternal hyperthermia at critical stages of neurulation.

2. MATERIALS AND METHODS

2.1 Materials
Adult female TO mice, about 30 gm in weight and about 6 weeks of age were mated with males of the same
stock in the evening and vaginal plugs identified in the following morning were taken to indicate successful
mating. Plug positive day was be day 0 of gestation. A minimum of 10 mice formed an experimental
group. All animals were kept under 12:12 hour light: dark cycles, in rooms maintained at a temperature of
21±1°C. They had free access to water and a laboratory chow provided ad libitum. Our previous

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experiments had shown that TO strain mice were sensitive to NTD when the experimental interference with
neural tube closure on gestation day (GD) 8.
2.2 Methods
2.2.1 Experiment 1
Mice were exposed on GD 8 to a single dose of heat for one hour in a water bath maintained at 43° C. This
treatment based on our rat experiments (Padmanabhanr et al., 2005) resulted in extreme maternal mortality.
2.2.2 Experiment 2
We then subjected mice to progressively decreasing periods of time to the same temperature and observed
that it was only a single exposure for a 12 minute period resulted in minimal maternal mortality, but there
was only a 3% incidence of exencephaly.
2.2.3 Experiment 3
We therefore treated groups of mice on GD 8 to a single or two successive doses of heat at 3 hour intervals
for 9 minutes. This treatment procedure resulted in NTD and fetal growth retardation. The controls were
sham treated in the water bath maintained at 37.5º C.
2.2.4 End points
All animals were killed on GD 18 by cervical dislocation and the total number of implants, resorptions and
living fetuses were counted. Live fetuses were blotted dry and weighed individually and fixed in 95%
ethanol solution. The incidence of exencephaly and other external and internal malformations were
recorded according to Sterz and Lehmann [20] slightly modified in our laboratory. The modification
comprised sophistication in surgery whereby the embryonic skeleton could be studied after visceral
examination. The skeletal defect s will be examined after staining with Alcian blue and alizarin red-S [21].
2.2.5 Statistics:
The data presented here came from Experiment 3 which comprised 57 mice of which 27 were controls.
ANOVA was used for statistical analysis. P<0.05 was regarded significant.
2.2.6 Animal Ethics:
FMHS Animal Research Ethics Committee approved this project and the experiments were conducted
according to the Ethical codes laid down by this Committee.

3. RESULTS
Maternal sham treatment on GD 8 was found to result in a 2-3% incidence of embryonic resorption, which
was not different from that of the non-treated control recorded in our previous experiments. There was no
fetal growth retardation or any congenital malformation. The embryonic resorption in the single and double
dose hyperthermia groups were 21% and 28% respectively. The weights of fetuses of these treatment
groups were only 75% of the corresponding controls. Whereas intrauterine growth retardation (IUGR) in
terms of reduction in mean fetal body weight was not dose-dependent, about 10% more of double dose
group fetuses were at -2SD level of IUGR.

A single dose of heat exposure resulted in an incidence of exencephaly in about 16% of live fetuses. The
most interesting finding was a threefold increase in the incidence of exencephaly when a second dose of
heat exposure occurred to mice on GD 8. Thus, it is obvious that repeated exposure to heat has an additive
effect in terms of embryonic resorption and exencephaly incidence.

The exencephalic embryos, irrespective of the heat -dose were all growth retarded. There was a
preponderance of female embryos involved in this major malformation syndrome characterized by IUGR,
open and everted cranial neural folds which were hemorrhagic, maxillary and mandibular hypoplasia, low-
set microtia, protruding tongue, polyhydramnios and severe axial skeletal malformations. The skull vault
was missing and basicranial bones were crowded resulting in a significant reduction in cranial diameters.

4. DISCUSSION
Anencephaly, encephalocele and spina bifida are often grouped together and referred to as neural tube
defects (NTD) in clinical practice. NTD affect approximately 0.1% liveborn infants. Although not clearly
established, NTD are reported to be of multifactorial inheritance. They have both a genetic and an
environmental component in their inheritance. This concept led us to believe that gene-teratogen
interactions might be important in the etiology of NTD. One way of approaching this issue is to employ
animal models, which have a low background frequency of NTD and determine if low or sub teratogenic
doses of known teratogens would amplify this incidence. We have previously shown the susceptibility
cofactor in TO mouse interacts with aspirin, ethanol, valproic acid, and all trans-retinoic acid [15-191]. In
the present study, we wanted to determine if this held true for a physical agent such as hyperthermia. The

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data presented here show that hyperthermia does induce NTD dose-dependently and that the NTD fetuses
present a syndrome of malformations.

Finnell et al [22] reported several years ago that hyperthermia-induced NTD in mice was strain-dependent.
They compared response of SWV, LM/Bc, SWR/J, C57BL/6J, and DBA/2J strains to maternal
hyperthermia in terms of exencephaly and observed a hierarchy of susceptibility among them [22]. Our
data indicate the susceptibility of hitherto unexplored TO strain-susceptibility to hyperthermia. These data
also suggest that the same susceptibility cofactor may respond to a variety of chemical and physical agents.
Thus, it appears that the susceptibility factor is rather nonspecific to noxious agents during the window of
neurulation period in the mouse.

Maternal exposure to elevated temperatures during pregnancy has been shown to be teratogenic to animal
and human embryos. In fact, hyperthermia has the dubious credit of being discovered as a teratogen in
laboratory animals and subsequently in humans. Our preliminary electron microscopic studies on heat-
treated rodent embryos indicate a plethora of cellular and subcellular morphologic alterations including
apoptosis in the neuroepithelium and head mesenchyme. Thus it appears that heat –treated embryos not
only exhibit expression of inducible and constitutive species of heat shock proteins and altered gene
expression but also significant morphologic changes that might be the underlying mechanisms of heat-
induced congenital malformations in laboratory animals.

CONCLUSION
Exposure of TO strain of mice on GD 8 to single and two successive doses of hyperthermia results in dose-
dependent increase in incidence of NTD and axial skeletal defects indicating this model of NTD could be
used in further investigation of pathogenetic mechanisms of neural tube anomalies .

ACKNOWLEDGEMENT
This work was supported by an Individual Grant (02-13-8-11/03 from Research Affairs of the
U.A.E.University.

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