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Effect of estradiol and soy extract on the onset of ptz-induced seizure in ovariectomized rats: implications for nurses and midwives

Nurs Midwifery Stud. In Press(In Press):e33428.
Published online 2016 February 14.
Effect of Estradiol and Soy Extract on the Onset of PTZ-Induced Seizurein Ovariectomized Rats: Implications for Nurses and Midwives Nastaran Afsordeh,1 Azhdar Heydari,1 Mahmoud Salami,1 Samaneh Sadat Alavi,1 Elaheh Arbabi,1 SaraKarimi,1 and Gholamali Hamidi1,* 1 Physiology Research Center, Kashan University of Medical Sciences, Kashan, IR Iran * Corresponding author: Gholamali Hamidi, Physiology Research Center, Kashan University of Medical Sciences, Kashan, IR Iran. Tel: +98-3155542999, Fax: +98-3155575057,E-mail: [email protected] Received 2015 September 26; Revised 2016 January 09; Accepted 2016 January 10.
Background: In many epileptic women, the ovarian cycle influences the risk and severity of seizures. Previous studies have reported
inconsistent findings regarding the neural excitatory effects of estrogen. Soybean is a rich source of plant estrogens. Therefore, soy
intake can affect the risk and severity of seizures in women. Nurses and midwives need to be aware of the effects of diets containing
estrogen in pregnant and post-menopausal women, as well as during the sexual cycle periods.
Objectives: Due to the limitations of human studies, this study aimed to investigate the effect of estradiol and soy extract on the
onset of pentylenetetrazol (PTZ)-induced seizures in ovariectomized rats.
Materials and Methods: In this experimental study, sixty female Wistar rats were randomly divided into six groups of ten. All of
the groups except for the negative control groups were ovariectomized. One group received estradiol (100 µg/kg), one group was
treated with alcoholic extract of soy (20 mg/kg), and the other group received vehicle over 28 days. PTZ (90 mg/kg) was used to
induce seizure in all groups. Behavioral changes were observed over 30 minutes. All data were analyzed using a two-way analysis of
variance with Tukey's post-hoc test.
Results: The mean ± SD of the clonic seizure threshold was 61.84 ± 8.53 in the group that received estradiol, while it was 71.70 ±
10.85 in the control group, 64.69 ± 3.23 in rats with laparotomy without removing the ovaries, 51.30 ± 6.84 in rats with laparotomy
and removing the ovaries, 52.7 ± 7.18 in ovariectomized rats that received sesame oil as vehicle, and 59.2 ± 4.91, in the soy groups.
Therefore, estradiol significantly increased the clonic seizure threshold, while the alcoholic extract of soy had no effect.
Conclusions: We concluded that the chronic administration of estradiol has an anticonvulsant effect in the animal model. However,
alcoholic extract of soy has no effect on the onset of clonic seizures.
Keywords: Soybean, Estradiol, Ovariectomized, Seizure to seizure. This ratio is reduced during the menstrual cycle,and it is increased during the early and middle luteal cycle Sex steroids such as estrogen have regulatory effects Changes in seizure frequency among men and women on neuronal excitability The frequency and severity of may occur in cycles of eight to 46 days and, interestingly, seizures can be altered by changes in sex hormones and these changes sometimes occurs in cycles of 28 days, which they can be worse during certain phases of the menstrual is similar to the female sexual cycle cycle Catamenial epilepsy is a subset of epilepsy thatincludes women whose seizures are exacerbated by their Women with epilepsy receive antiepileptic drugs such menstrual cycle The prevalence of epilepsy is 50 in as carbamazepine, oxcarbazepine, and phenobarbital.
100,000 in developed countries Due to the effects of These medications increase the risk of osteoporosis sex hormones, for many decades it was believed that there The estrogen level will decrease following ovariectomy and is a direct relationship between estrogen levels and seizure menopause, which can result in hot flashes and osteoporo- and that estrogen induces excitatory actions in the cen- sis. Hormone replacement therapy (HRT) is usually used tral nervous system However, various studies have sug- to relieve such symptoms Studies have shown that the gested both proconvulsant and anticonvulsant roles type of estrogen used in HRT is important, and that equine for estrogen.
conjugated estrogen might increase the risk and severity Almost 50% of women who experience convulsion are of childbearing age During ovulation, reducing the ra- A traditional food source in Asia for many years, soy- tio of progesterone to estrogen might predispose women bean is a rich source of phytoestrogens Phytoestro- Copyright 2016, Kashan University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial4.0 International License ( which permits copy and redistribute the material just in noncommercial usages, provided theoriginal work is properly cited.
Afsordeh N et al. gens contain isoflavones, coumestans, and lignans 70% ethanol. It was then kept at room temperature for 72 Isoflavones display similar properties to endogenous es- hours. Then, the extract was separated through the fun- trogens and they can bond with estrogen receptors nel. The dried extract was placed in water, alcohol, and However, the estrogenic activity of phytoestrogens is poor.
other additional material to evaporate for 16 hours. In or- Therefore, they are known as selective estrogen receptor der to produce the drug, a certain amount of powder was modulators These selective estrogen receptor modu- dissolved in water and a dose of 20 mg/kg was achieved.
lators act as agonists or antagonists in different tissues Nurses and midwifes play important roles not only in con- 3.3. Surgery and Treatments trolling seizures during pregnancy, but also in educatingand caring for women with convulsion. They should be The study was conducted at the physiology research aware of the latest knowledge concerning the treatment center, Kashan University of Medical Sciences. The female and prevention of convulsion in women in order to not rats were anaesthetized using an intraperitoneal injection only educate them, but also to monitor them and reduce of Ketamine 100 mg/kg/ + xylazine 10 mg/kg. Then, under the side effects of anticonvulsants. Given the side effects aseptic conditions, the researchers laparotomized and or of anticonvulsant drugs and HRT, as well as the effects of ovariectomized the relevant animals. Two days after recov- phytoestrogens and soybean on estrogen receptors, a ques- ery estradiol (100 µg/kg) was injected subcutaneously tion remains regarding whether soybean can be used as an and soy extract (20 mg/kg) was gavaged daily for 28 days in extrinsic source of estrogen without any associated estro- the appropriate groups.
genic side effects. If this were the case, then soy could beused to reduce the risk and severity of seizure in women.
3.4. Behavioral Study Pentylenetetrazole (Sigma Aldrich, St. Louis, Missouri, USA) was dissolved in sterile 0.9% saline to prepare a freshsolution. In all study groups, the animal model of seizure Due to the limitations of human studies, this study was achieved via the intraperitoneal injection of 90 mg/kg aimed to investigate the effects of the chronic admin- pentylenetetrazole. The rats were placed in a Plexiglas istration of estradiol and alcoholic extract of soy on cage following PTZ injection and they were observed for pentylenetetrazol (PTZ)-induced seizure in ovariectomized The seizure stages were classified as my- female rats.
oclonic twitch, clonic seizures, tonic hindlimb extension,and death. Although all of these stages were observed 3. Materials and Methods
and their times were recorded using a chronometer, onlythe clonic seizure threshold (i.e., the time until the start 3.1. Study Design and Animal Groups of clonic seizure) was used to compare between different In this experimental study, sixty female Wistar rats weighing 170 - 220 gr were studied. The animals werehoused in standard cages under standard temperature 3.5. Ethical Considerations (25°C) and humidity (50% - 60%) with a 12-hour light and The institutional review board and the ethics commit- dark cycle. The animals were fed with standard chow and tee of Kashan University of Medical Sciences approved this they had free access to water. The rats were randomly di- study. Food and water were provided to the animals ad li- vided into six groups of ten as follows: 1, CON (intact female bitum. All ethical considerations regarding the use of labo- rats); 2, SHAM (laparotomy in the female rats without re- ratory animals were observed in light of the requirements moving the ovaries); 3, OVX (laparotomy in the female rats of the Helsinki declaration.
accompanied by removal of the ovaries); 4, VEH (ovariec-tomized rats that received sesame oil as a vehicle); 5, EST(ovariectomized rats that received 100 µg/kg estradiol); 3.6. Statistical Analyses and 6, SOY (ovariectomized rats that received 20 mg/kg al- We analyzed the data using SPSS statistical software, coholic extract of soy).
version 13.0. All data were expressed as mean ± SD. The Kolmogorov-Smirnov test was used to assess the normality 3.2. Preparing the Materials of the data in each group. Differences between the onset To prepare the hydro alcoholic extract, 100 g of pow- of seizures were analyzed using two-way analysis of vari- dered soybean varieties (Glycine max, Barij Essence Phar- ance and Tukey post-hoc tests, and the difference was con- maceutical Co.) was prepared and dissolved in 800 mL of sidered statistically significant when P < 0.05.
Nurs Midwifery Stud. In Press(In Press):e33428.
Afsordeh N et al. 4. Results
a Kainic acid-induced seizure model after an injection of 0.1mg/kg of estradiol benzoate within 24 hours is neuropro- As shown in no significant difference was found tective in the stria terminalis Moreover, the anticon- between the SHAM group and the CON group in terms of vulsant effects of estradiol were shown in ovariectomized the onset of clonic seizure. In other words, laparotomy rats in models of picrotoxin- and cyclosporine- in- did not affect the onset of seizure. In addition, the onset duced seizures In the current study, the prolonged of clonic seizures in the VEH group was not significantly use of estradiol was found to delay the onset of seizures in different to that in the OVX group. In other words, use of ovariectomized rats. This finding was in line with a previ- the sesame oil vehicle had no effect on the onset of seizure.
ous study in which the prolonged usage of estrogen had However, the onset of clonic seizures in the OVX group sig- neuroprotective effects against neuronal damage-induced nificantly decreased compared to that in the SHAM group seizures However, another study reported that the (P < 0.002).
acute administration of estrogen significantly increases Moreover, the onset of clonic seizures increased signif- the risk of seizures icantly in the estradiol group compared to the VEH group The pharmacological levels of estradiol can reversibly (P < 0.019) In addition, the results of treatment decrease the N-Methyl-D-aspartic acid-induced flow, excita- with the soy extract did not show significant changes com- tion, and cell death of nerve cells The activation of glu- pared to the VEH group tamate or the inactivation of GABA plays an important rolein the process of creating epilepsy, In another study, Table 1. Mean and Standard Deviation in the CON, VEH, SHAM, and OVX Rat Groups
subcutaneously planted estradiol capsules were used to in Terms of the Onset of Clonic Seizures Induced by PTZ in Ovariectomized Ratsa treat PTZ-induced seizures and it was suggested that estra-diol may play the role of an anticonvulsant through 3α-5α- The Onset Of Clonic Seizures (Sec)
TPH, which increases in the hippocampus and functions as a GABAA receptor agonist The effects of estrogen on seizure depend on various factors such as duration of treat- ment, methods of administration, or the neurotransmit-ter in the nervous system that is involved in seizure The anticonvulsant effects of estrogen could possibly be at- Abbreviations: CON (intact female rats); OVX (laparotomy in the female rats ac- tributed to substances such as neuropeptide Y (NPY) companied by the removal of the ovaries: ovariectomized rats); SHAM (laparo-tomy in the female rats without removing the ovaries); VEH (ovariectomized which is a strong inhibitory neurotransmitter in the cen- rats that received sesame oil vehicle).
a tral region of the hippocampus Sex hormones might Values are expressed as mean ± SD.
b P < 0.002, as compared to the SHAM group.
act through different mechanisms such as changing thegene expression, releasing neurotransmitters, and a directeffect on neurotransmitter receptors In this study, 20 mg/kg of alcoholic extract of soy had Table 2. Mean and Standard Deviation After 28 Days' Administration of EST and SOY
on the Onset of Clonic Seizures Induced by PTZ in Ovariectomized Ratsa
no effect on the onset of seizures. However, Mohammad-pour et al. showed that the proconvulsion effect of soy The onset of clonic seizures (sec)
extract is dependent on the presence or absence of ovar- ian hormones Moreover, previous studies have shownthat soy extract can mimic the effects of estrogen and so reduce the latency of myoclonic and generalized tonic Abbreviations: EST (ovariectomized rats that received 100 µg/kg estradiol) and clonic seizures in ovariectomized rats However, in SOY (ovariectomized rats that received 20 mg/kg alcoholic extract of soy).
a Values are expressed as mean ± SD.
the present study we did not observe this effect. Nonethe- b P < 0.019, as compared to the VEH group less, earlier studies reported that phytoestrogens play ei-ther estrogen or estrogen antagonist roles Soy con-tains the isoflavones genistein and daidzein Westmark et al. showed that the isoflavones in soybeans are effec-tive in decreasing the threshold of seizure in mouse mod- The present study showed that ovariectomy decreased els and, therefore, they may increase the risk of epilepsy the onset of PTZ-induced seizure. On the other hand, estra- Westmark's hypothesis declares that the soy phytoe- diol displayed anticonvulsant effects. The protective ef- strogens interact with the metabotropic glutamate recep- fects of estrogen against epileptic activities have also been tor and stimulate the production of the synaptic proteins reported in previous studies. Pereno et al. represented that that play a key role in reducing the seizure threshold Nurs Midwifery Stud. In Press(In Press):e33428.
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