Roles Of Estrogen In Ovariectomized Rats With Seizure Induced By Kainic Acid

Epilepsy is one of common diseases in nervous system. Seizure frequency was related to menstrual cycle in female patients, and the seizure threshold at proestrus of estrous cycle was the lowest in female rats due to a high level of estrogen and/or a low level of progestin.The antiepileptic effects of progestin were almost defined in the studies on clinic and animal models, but the effects of estrogen on epilepsy were still argued. In our early study, it was showed that high dose of estrogen can accelerate the seizure of ovariectomized (Ovx) rats. Up to now, however, no systematic study was reported about how to define the high or low dose of estrogen and what events were happened in seizured rats with pretreated by estrogen in different doses? Therefore, in the present study, on the basis of the doses of estrogen objectively defined, effects of estrogen pretreatment on Ovx rats' seizure induced by kainic acid were investigated. The latency and severity of seizure was used to evaluate the behaviors of animal, and the injury degree of hippocampus subfields was indicated by cell counts. Serum E₂ level, hippocampal amino acids neurotransmitters and estrogen receptor expression were used as biochemical index for assessing the roles of estrogen in seizure.The etiology and contributing factors of epilepsy were extremely complex. If the precondition of estrogen really affected the seizure of rats, it is certainly for us to attempt to understand which genes were involved in the estrogen affecting seizure and which functions were contributed to these genes?This project was consisted of three parts, and the main aim was to explore the effects of estrogen on seizure.Part I. Preparation of Seizure Model of Ovariectomized Rats Pretreated with Estrogen and Behaviors Testing.Objective: (1) To determine the levels of serum gonadal hormones in the ovariectomized rats (Ovx rats) with and without treated by using various dosesestrogen at different time points, and to select typical high and low doses of estrogen precondition in the pre-seizure rats. (2) To evaluate the reasonable time points for inducing seizure to the Ovx rats with administrating estrogen, and to define the effects of estrogen precondition on behaviors of seizure rats. Methods: Vaginal cytological characteristics of normal cycle and Ovx rats were determined by observing vaginal smear under light microscopy. The levels of serum estradiol (E₂), progesterone (P) and testosterone (T) in rats which include proestrus, Ovx7d, Ovx13d and 5d after EB treated with different doses (25ug/kg, 1, 2, 4, 8 and 20 mg/kg) were measured by the method of electrochemical illusion. The Ovx13d rats were injected kainic acid (KA, 5mg/kg) intra-tail vein to induce seizure. The latency and severity were measured following KA administration, according to the Racine scale. Results: (1) Only a bit of vaginal epithelial cells were observed from the vaginal smears of Ovx rats. The serum hormonal levels, including E₂, P and T were obviously declined in sera of Ovx rats compared to the proestrus rats. (2) It was two reasonable time points of Ovx7d for precondition of exogenous EB to Ovx rats and Ovx13d for injecting KA to induce seizure, because of serum E₂ levels no significant difference between Ovx7d and Ovx13d groups, but a decreased P level and increased the ratio of E/P in Ovx13d group. (3) The levels of E₂, P and T in sera of E25ug/kg group were distinctly lower than that in sera of proestrus rats, but P level in former was higher than that in Ovx rats. The levels of E₂ in Ovx rats increased with administrating EB, and the highest dose of EB had to be limited in 2mg/kg, otherwise the level of serum E₂ will be exceed detectable scope. In addition, there was a significant difference in the levels of serum E₂ between E25ug/kg and E2mg/kg groups, but were not in the levels of P and T. Therefore, the doses of 25ug/kg and 2mg/kg of E₂ were suitable to evaluate the effects of E₂ on seizure alone. (3) The latency of minor seizure state could be shorten by pretreated with both 25ug/kg and 2mg/kg of EB, however, latency of severe seizure state was shortened only at high dose. Conclusions: The results of cytological characteristics and gonadal hormones levels demonstrated that the ovariectomy was extremely successful. The precondition of 25ug/kg and 2mg/kg of EB were typical high and low doses used in the pre- seizure Ovx rats. The time points of Ovx7d and Ovx13d were reasonable for administrating EB and injecting KA, respectively. Both 25ug/kgand 2mg/kg of EB could shorten the latency of seizure kindling, however, only a higher dose could accelerate epileptic-like discharge to spread in brain.Part II. Effects of Estrogen Preconditioned on Hippocampus Related to Seizures Induced by Kainic Acid in Ovariectomized Rats.?Section I. Effects of Estrogen Preconditioned on Hippocampal Neurons Injury in Kainic Acid-Induced Seizure Ovx Rats. Objective: To compare effects of estrogen precondition in different doses on cell lose of hippocampal subfields in seizured rats, and to explore the roles of estrogen in seizure.Methods: The Ovx rats were randomly divided into nine groups ,ie, Ovx, Ovx+EB25ug/kg, Ovx+EB2mg/kg, Ovx+KA (minor/severe), Ovx+EB 25ug/kg +KA (minor/severe) and Ovx+EB2mg/kg+KA (minor/severe) groups. NeuN positive cells stained by immuno-fluoresent histchemical method were used as indicators of surviving neurons. Cells count and data analysis were conducted by using Image-Pro Plus 5.1 and SPSS11.5 softwares. Results: 1) No significant effects were observed in the numbers of NeuN positive cells/length of hippocampal subfields comparing the preseizure rats with and without pretreated by EB. 2) The lose of NeuN positive cells in the hippocampal subfields sensitive to injury was resulted from seizure, which was related to severity of seizure, no matter whether the seizured rats were pretreated with EB. 3) Both low and high doses of EB could relieve the loss of NeuN positive cells in hippocampus of seizured rats. Conclusions: 1) Seizure was the primary cause of hippocampal neurons death which was related to severity of seizure. 2) EB at both low and high doses can relieve the damage of hippocampal subfields sensitive to injury, although they cannot completely erase the damage from seizure.Section II. Effects of Estrogen Preconditioned on Levels of Hippocampal Amino Acid Neurotransmitters in Kainic Acid-Induced Seizure Ovx Rats.Objective: To analyze the changes of hippocampal amino acid neurotransmitters in various doses of extrogen preconditioned on ovariectomized (Ovx) rats with or without seizure induced by kainic acid, and to find out the relationship of amino acid neurotransmitters changes and E or seizure, and to explore the roles of estrogen in seizure. Methods: The Ovx rats were randomly divided into nine groups ,ie, Ovx, Ovx+EB25ug/kg, Ovx+EB2mg/kg, Ovx+KA (minor/severe), Ovx+EB25ug/kg +KA( minor /severe) and Ovx+EB2mg/kg+KA (minor/severe) groups. Exciting (Glu and Asp) and inhibitory amino acids (Gly and GABA) in hippocampus from different groups were measured by HPLC method. Results: 1) Both Glu and Asp levels were declined in hippocampus of Ovx rats pretreated with high and low doses EB before seizure, but no significant changes in the levels of Gly and GABA. 2) Seizure induced GABA and Gly to quicken synthesis in hippocampus, no matter whether the rats with and without pretreated by EB. 3) The precondition of EB could affect the changes of hippocampal amino acid neurotransmitters on both pre-seizure and seizure. The effects on the rats of pre-seizure, EB mainly decreased the levels of exciting amino acids. The effects on the seizure, EB could induce the synthesis of exciting amino acids, and more importantly it induced the synthesis of inhibitory amino acids.Conclusions: Seizure was a primary cause for inducing the elevation of GABA and Gly at the early stage of hippocampus injury. EB had different effects in changing hippocampal amino acid neurotransmitters on the pre-seizure and seizured rats. A lower dose of EB may protect the hippocampus damage from the seizures. Section III. Changes of Serum Estradiol levels and Expressions of Hippocampal Estrogen Receptor β (ERβ) in Estrogen Preconditioned Ovariectomized Rats with before& after Seizures Induced by Kainic acid. Objective: To analyze the changes of serum estradiol level and hippocampalestrogen receptor β ( ERβ) expression in various doses of estrogen preconditioned ovariectomized (Ovx) rats with or without seizure, and to determine the relationship among estrogen, ERβ and seizure. Methods: The Ovx rats were randomly divided into Ovx group, various doses of EB-pretreated groups (Ovx +EB groups), seizure groups (Ovx +KA groups) and various doses of EB-preconditioned seizure groups (Ovx +EB+KA groups). The serum estradiol level and hippocampal ERβ expression were detected by electrochemical illusion and immuno-Western blot, respectively. Results: 1) Serum estradiol levels in Ovx rats were elevated with the increasing doses of EB pretreatment(≥EB 1mg/kg), except that EB25ug/kg group (88.8 ±3.23 pmol/L) was no significant difference compared with Ovx group (88.49±8.31 pmol/L). However, the EB preconditioned did not affect the ERβ expression in hippocampus of Ovx rats. 2) At the early stage of seizure, serum estradiol level and hippocampal ERβ expression were significantly decreased, but the latter were not related to the degree of seizure.3) The levels of ERβ in hippocampus of both pre-seizured and seizured rats with pretreated by low dose of estrogen were not any significant differences (p>0.05), so the estrogen preconditioned in a low dose could efficiently inhibit ERβ decrease resulted from seizure.Conclusions: The hippocampal ERβ expression in Ovx rats was correlated with seizure. Low dose of estrogen could efficiently inhibit ERβ decrease resulted from seizure. Part III. Effects of Estrogen Preconditioned on Hippocampal Gene Expression after Seizure Induced by Kainic acid in Ovariectomized Rats.Objective: To analyze the hippocampal gene expression profiles of ovariectomized (Ovx) rats with preconditioned estrogen on seizure induced by Kainic acid, and to explore effects of estrogen on hippocampal of seizure rats. Methods: The patterns of hippocampal gene expression from EB-preconditioned Ovx rats with or without seizure induced by kainic acid (KA)were obtained by using 10,000-gene microarray assay. The functional cluster analysis was used for screening the functional groups of dramaticallyaltered genes.Results: cDNA microarray assay showed that 392 genes were identified to besignificantly differential expressions, the known functional genes were 151.The down-regulated and the up-regulated functional genes were 131 and 21,respectively. These genes were classified into 8 functional groups, 5down-regulated groups (containing 21 genes) were involved in apoptosis,anti-apoptosis, neurogenesis and long term potential (LTP); 3 up-regulatedgroups (containing 4 genes ) were involved in signal transduction related withcellular surface receptors, electron transport.Conclusions: EB could reverse the hippocampal gene expressions of Ovxrats with seizure, of which one main role maybe promote neuronal apoptosis.