| Epilepsy is a common disease and the detail mechanism of this disease is still unknown. In clinical, some women patients who have epilepsy have some special characters. Their symptoms have a tight relationship with their menstrual cycle. In special menstrual cycle, symptoms of epilepsy will aggravate and drug control will decrease. We call this kind of epilepsy as menstrual epilepsy. Estradiol as an important sexual hormonal of women may play a key role in this kind of epilepsy. But the effect of estradiol is conflict. Some studies show it has anti-convulsant effect and some studies show it has pro-convulsant effect. We hypothesis that estradiol may have bilateral effect on epilepsy and the dose of estradiol may be play a key role. In this study, we use cultured neurons to study the effect of estradiol. Phenol red as a PH indicator can mimic the bio-effect of estradiol. Whether it has effect on neuron eclectic activities and which dose is the perfect dose for neuron culture? We try to research the effect of estradiol in epilepsy, whether it has a bilateral effect on epilepsy and has a relationship with dose and the effect of sub-receptors.Part I Improvement the dose of phenol red a PH indicator in culture mediumObjective:To study the effect of phenol red on neurons abnormal eclectic activities. Methods:Use different doses of phenol red (0,10,21.5,30,60,100,200uM) to co-culture with neurons for14days.30μM phenol red and ICI182780co-culture with neurons for14days. Record neurons action potentials using patch clamp. Results:The percentage of neurons abnormal eclectic activities are OμM (84%,16/19);10μM (60%,12/20);21.5μM (21%,5/24);30μM (9.5%,2/21);60μM (43%,3/7),100μM (53%,9/17);200μM (83%,10/12). The percentage of neurons abnormal eclectic activities of30μM is lowest than that of0、10、60、100、200μM(p<0.01). The frequency of neurons abnormal eclectic activities are OμM(0.024±0.005Hz, n=19);10μM(0.006±0.002Hz, n=20);21.5μM(0.007±0.003Hz, n=24);30μM(0.001±0.001Hz, n=21);60μM(0.015±0.008Hz, n=7);100μM(0.01±0.004Hz, n=17);200μM(0.028±0.011Hz, n=12). The frequency of30μM is lowest than that of others (p<0.01). When30μM phenol red co-culture with ICI182780, the percentage and frequency of neurons abnormal eclectic activities of30μM+ICI182780(80%;0.024±0.006Hz) are higher than that of30μM(9.5%;0.001±0.001Hz)(p<0.01). Conclusions:Phenol red has a dose-dependent effect on neurons abnormal eclectic activities and30μM phenol red suppress abnormal activities through estrogen receptors.Part II The effects of estradiol and its sub-receptors on neuron abnormal eclectic activity in cultured neuronsObjective:Study the effect of estradiol on neuron abnormal eclectic activity in cultured neurons. Methods:1. Take different doses of estradiol(0.1,0.3,1ng/ml) and alcohol(0.002%) as control to co-culture with neurons for3days after14days culture. Record neurons action potentials using patch clamp.2. At the first day of culture, using different doses of estradiol(0.03,0.1,0.3,1ng/ml)and alcohol(0.002%) as control co-culture with neurons for14days. During incubation, keep the stable level of estradiol and alcohol. After14days, we record the eclectic activities of neurons.3. At the first day of culture, using0.1ng/ml estradiol and different doses of MPP-D(ERα antagonist)(100,10,1nM) and PHTPP(ERβ antagonist)(100,10,1nM) co-culture with neurons. Use patch clamp to record neurons eclectic activities after14days.4. At the first day of culture, using lng/ml estradiol and MPP-D(100nM) and PHTPP(100nM) co-culture with neurons. Use patch clamp to record neurons eclectic activities after14days. Results:1. Neurons cultured for14days, co-culture with alcohol and different doses of estradiol for3days. The percentage of neurons with abnormal activity:alcohol (73%,22/30); estradiol0.1ng/ml(13%,3/23);0.3ng/ml (37%,11/30); lng/ml (65%,13/20). Compared to alcohol and estradiol lng/ml,0.1ng/ml estradiol obviously suppress abnormal activities (p<0.01). The frequency of abnormal activities:alcohol (0.019±0.005Hz, n=30); estradiol0.1ng/ml(0.007±0.007Hz, n=23);0.3ng/ml (0.0079±0.003Hz, n=30);1ng/ml (0.037±0.016Hz, n=20). Compared to alcohol and estradiol lng/ml,0.1ng/ml estradiol obviously suppress the frequency of abnormal activities (p<0.01).2. Alcohol and different doses of estradiol culture with neurons for14days. The percentage of neurons with abnormal activity:alcohol(80%,12/15); estradiol0.03ng/ml(47%,8/17);0.1ng/ml(24%,4/17);0.3ng/ml(56%,15/27);1ng/ml (94%,15/16).Compared to alcohol and other dose of estradiol, O.lng/ml estradiol obviously suppress abnormal activities (p<0.01). The frequency of abnormal activities:alcohol (0.016±0.003Hz, n=15); estradiol0.03ng/ml(0.04±0.018Hz, n=17);0.1ng/ml(0.004±0.002Hz, n=17); 0.3ng/ml (0.01±0.004Hz, n=27);1ng/ml (0.034±0.007Hz, n=16).Compared to alcohol and other doses of estradiol,0.1ng/ml estradiol obviously suppress the frequency of abnormal activities (p<.0.01).3. Estradiol0. lng/ml with different dose of MPP-D (100,10,1nM) and PHTPP(100,10,1nM) co-culture with neurons. The results show that estradiol0.lng/ml with MPP-D and PHTPP100nM, the percentage and frequency of abnormal eclectic activity of MPP-D (80%,0.046±0.011Hz)and PHTPP(89%,0.069±0.016Hz) are no difference. But they are higher than that of0.lng/ml estradiol(24%,0.004±0.002Hz)(p<0.01). When the dose of MPP-D and PHTPP is lOnM, there is no difference between MPP-D(14%,0.004±0.004Hz) and estradiol(24%,0.004±0.002Hz).The percentage and frequency of PHTPP (83%,0.025±0.009Hz) are higher than that of estradiol (p<0.01). When the dose of MPP-D and PHTPP is1nM, there is no difference between MPP-D(11%,0.003±0.002Hz) and estradiol (24%,0.004±0.002Hz).The percentage and frequency of PHTPP (67%,0.052±0.029Hz) are higher than that of estradiol (p<0.01).4. Estradiol lng/ml with MPP-D (100nM) and PHTPP (100nM) co-culture with neurons. The percentage and frequency of MPP-D(20%,0.003±0.002Hz)are lower than that of estradiol(94%,0.034±0.007Hz)(p<0.01).There is no difference between PHTPP(80%,0.04±0.015Hz) and estradiol(94%,0.034±0.007Hz). Conclusions:Estradiol has a dose-dependent effect on neurons abnormal activities. It suppresses neurons abnormal eclectic activities,when its dose changed it appears to promote neurons abnormal eclectic activities. The0.lng/ml dose maybe the reversal point of the dramatic change. The results of antagonist also show that estradiol probably promote abnormal activities through ERa and suppress abnormal activities of cultured neurons through ERβ.Part III The effects of estradiol in epileptic animal modelObjective:Study the effect of estradiol in epilepsy animals. Methods:1. Adult female SD rats with at least two normal4-day estrous cycles were prepared for ovariectomy. After7days of ovariectomy, the rats were buried with micro-pipe at their lateral ventricle. After7days of buried pipe, the rats were grouped into alcohol team(giving alcohol as control for3days) and estradiol team(estradiol0.75ng for3days) at random. Male SD rats and female SD rats with estrus were grouped, too. All rats induced epilepsy by PTZ(40mg/kg). All rats praxiology were standardized following Racine code.2. After lateral ventricle buried pipe female SD rats were grouped into MPP-D team and PHTPP team at random. Inject MPP-D and PHTPP100nM through the pipe at least3days before epilepsy induced. PTZ(40mg/kg) were given to induce epilepsy when female rats were in estrus. Results:Study the score by Racine code of male rats, ovariectomy rats, estrus rats and ovariectomy rats with estradiol(0.75ng) are3.5±1.0(n=10),0.143±0.143(n=7),5.4±1.0(n=9),4.7±0.8(n=7). The score of ovariectomy rats is lowest than that of others (p<0.01).2.The score of estrus rats,estrus rats with MPP-D and estrus rats with PHTPP are5.4±1.0(n=9),1.9±1.1(n=8),3.1±1(n=8). The score of estrus rats with MPP-D is lower than that of estrus rats (p<0.05). Conclusions:Animals studies show that estradiol has effect on epilepsy and suppress epilepsy through ERβ.Conclusions1. Phenol red has a dose-dependent effect on neurons eclectic activities. Estrogen receptors may contribute to suppression of phenol red. The dose of phenol red in commercial culture medium is not suitable.2. Estradiol has a dose-dependent effect on cultured neurons abnormal eclectic activities whether for3days or14days.3. Estradiol sub-receptors have different effect on neurons eclectic activities. ERa may contribute to enhancement neurons abnormal activities and ERβ may contribute to suppression.4. Animal studies also show that estradiol has an effect on epilepsy and may suppress epilepsy through ERβ. |
PDF Full Text Request