Abstract
Topiramate is commonly used for
treating epilepsy in both children and adults. Recent clinical data suggests
that administration of topiramate to women during pregnancy increases the risk
of oral clefts in their offspring. To better understand the effects of
topiramate, an adult female zebrafish was dosed with topiramate, and the altered
morphologies were investigated in the adult females and their off springs. It
showed that topiramate-treated female fish had reduced oocyte maturation, and
the survival rates of their offspring were seriously decreased during
embryogenesis, in addition to cartilage malformation in some offsprings.
Therefore, topiramate causes both impairment of mother oogenesis, and bone
dysplasia.
Introduction
Topiramate is used for treatment of
migraine prophylaxis and Lennox-Gustuat syndrome. Topiramate is derived from
naturally occurring D-fructose, and have great effect in blocking the spread of
seizures. It is classified as Pregnancy Category D according to the FDA drug
grading that indicates positive evidence of human fetal risk. According to some
studies, the exposure of topiramate during the first trimester of pregnancy,
leads to an increase in the oral cleft rate in infants. However, variation in
the given doses, changes the result in both the mother and the offsprings.
Moreover, zebrafish was used as an animal model, since that it embryos are
easily collected, they have rapid development, and they have genetic similarity
to humans. In this study, it is noticed that after treatment with topiramate,
the reproductive ability of the mother was decreased, and there is a connection
between the maternal passages of the topiramate effect in offspring.
Materials and
Methods
1- Zebrafish maintenance:
first, maintain AB zebrafish stain under 14 h
on/10 h off light cycle. Then collect embryos and culture them in petri dishes
containing E3 water (5.0mM NaCl,0.17mM KCl,0.33mM CaCl2 and 0.33mM MgSO4) at 28
C, finally, add 1-phenyl 2 –thiourea during embryogenesis to inhibit pigment
formation.
2- Topiramate
treatment
Choose adult female fish (4 months
old) that had reached sexual maturity to undergo the topiramate containing
diet.Mate the females with males to completely ovulate eggs from their ovaries
to begin new cycle of oogenesis. Now start the dosage process by feeding fish
0.5 mg/g/day of topiramate- blended dry food once per day for 7 days. At the
end of the final day, breeding environment is set up, then collect embryos in
the following day morning.
Figure 1. Experimental design scheme
of topiramate treatment in Zebrafish
3- Preparation
and Staining of Paraffin Section
Fix the whole ovarian tissues in 4% Paraformaldehyde.
Then, rinse them in running water. On a microtome section the paraffin embedded
blocks at a thickness of 5 µm. Deparaffinize the slides in 55 C water bath for
10 minutes. After that immerse them in xylene two times for 10 minutes and in
ethanol in different concentrations for 5 minutes to rehydrate the tissues,
finally, rinse them in distilled water before staining.
4- Hematoxylin
and Eosin Staining
Stain the previous slides in
hematoxylin for 3-5 minutes, and wash them in running water until sections turn
blue. Stain them in 1% eosin Y for 10 minutes, then dehydrate the slides in
80%, 90%, 100% ethanol for 20 seconds each, then observe them under the
microscope.
5- Tissue
staining
Rinse the Paraformaldehyde-fixed
embryos with 70% ethanol. Stain them with alcian blue solution, then rehydrate
them with ethanol/H2O series (80%, 50%, 20%, and 0% ethanol). After that, rinse
them with phosphate-buffered saline (PBS) for 15 min. Then, rinse them with
trypsin. Remove excessive dye with 1% KOH/3% H2O2 (v/v) until conformation of
cartilage was fully observed.
Note: For vertebrate skeletal observation, alizarin red staining was
performed. Using the same fixation procedure as described above, specimens were
stained with 0.1 mg/mL alizarin red in 0.5% (w/w) KOH at room temperature and distained
with methanol.
6-
Microinjection
First, inject topiramate into the
embryo during 1-to 4 cell stage. Second, Inject 0.5, 1. 2 ng of topiramate
between the boarder of the cell and the yolk of the embryo. Third, raise the
injected embryos and analyze them.
Results
1- Topiramate Showed No Effect
on Weight of Experimental Female Fish.
For the
experimental group:
Its average weight was 0.88g (range0.7–1.1g)
at the beginning of the experiment which increased to 0.96g (range 0.9–1.1 g)
after treatment.
For the control
group:
Its average weight was also
0.88(range 0.7-1.1g) at the beginning of the experiment which increased to 1.05
(range 1.0–1.1 g).
The experimental group weight
increased 0.08g, while the control group weight increased 0.2 g.
Therefore, there is no significant
statistical difference when comparing the weight of the two groups.
2- Topiramate
Impairs the Maturation of Oogenesis
For the control
group:
A large portion of ovarian tissue
was filled with mature oocytes.
For the experimental
group:
There is a significant decrease in
the % of mature oocytes at both stages primary growth (I) and cortical alveolus
(II), only 40 % of the female fish were able to lay eggs.
This means that the uptake of
topiramate in female fish affects oogenesis and suggested that it may lead to
abnormal embryogenesis in offspring.
3-Effect of
Topiramate on Cartilage Development in Offspring
Compared to the control group, the
experimental group showed a lack or shortage of cranial and pharyngeal
development.In the experimental group, the ventral view showed a transformation
in Meckel`s cartilage, ceratohyal, and ethmoid plate.The lateral view showed
that the ceratobranchial was severely underdeveloped.Even that different individuals
causes divergent degrees of drug response, it is observed a similar
under-developed status in the craniofacial cartilage region in diverse
topiramte- treated offspring, and some of them have complete cartilage
development.Along the offspring's, only 23% showed cartilage abnormality.
Thus, it is
suggested that topiramate had a serious impact on cartilage development;
teratogenic factors were indeed passed from mother to offspring.
4- Craniofacial
Malformations on Topiramate-Treated Offspring
Topiramate-treated
mother fish showed the following, significantly
decreased L/W within the craniofacial region. Ossification reduction in the
post-cranial axial skeleton. Impaired ossification in the regions of ceratohyal
and Meckel’s cartilage.Decreased number of spinal columns from 13.2 to 6.2 was
shown in the treated offspring.
So, not only
cartilage development, but also bone formation was impaired by topiramate
treatment.
5- Topiramate
Affects Epiboly Progression of Offspring Fish
Embryos from topiramate- treated
fish showed a lower survival rate than usual.An average of 16.3 ± 15.6% of
early developmental malfunction, including aberrant epiboly migration at the
5.3 hpf stage, and failure to differentiate was detected at 8 hpf embryos. The
embryo was able to initiate epiboly progress with blastoderm formed, but the
enveloping layer of cells failed to migrate to the vegetal pole and accumulated
at the animal pole, forming a bubble like shape. The blastoderm was unable to
cover the yolk cell and halted before 50% coverage.As we increase topiramate
dosage, we observe that the survival rate decreases.
Therefore, topiramate
is transmittal, it causes epiboly deficiency.
Discussion
The reduction of weight and skeletal
development were observed side effects of topiramate with maternal toxicity. To
know the topiramate effect direct injection into embryos was done, the dosages
was calculated based on the assumption of absorption per clutch of eggs from
topiramate-treated mother fish. Precise calculation was ensured so as not to
overdose the fish and cause artificial toxicity and lead to experimental bias.
Therefore, based on this assumption, the teratogenic effects are due to
topiramate treatment.It is possible that infertility occurs in women who have
epilepsy and this may be due to the side effects of antiepileptic
medication.The fertility rate in women with epilepsy has been reported to be
lower compared with their nonepileptic female siblings. In fact, only 40% of
maternal fish were fertile in our study.Craniofacial abnormality is one of the
most common congenital birth defects, which affects the development of the
head, face, and neck. Thus In this study,the ratio of zebrafish with cartilage
malformation was about 23%, which is much higher than shown in a clinical study
on humans.
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