Effects Of Low Dose Estrogen On The Behavior, Hippocampal Neurodegeneration And Inflammation In Epileptic Rats

Estrogen is known to participate in the protection of a growing number of acute and chronic neurological disorders. Our previous experiments demonstrated that pretreatment with estrodiol benzoate attenuated insults in the areas of CA1, CA3 and hilus regions after the long-lasting severe seizures induced by intravenous injection of kanic acid. This phenomenon was also reported by the experiments in the model of pilocarpine-induced status epilepticus (Pilo-SE). Most of the former research had attributed this protective effect of estrogen to its neurotrophic and antiapoptotic functions, however, the anti-inflammatory impacts of estrogen on the epilepsy were seldom studied.A rapid-onset inflammatory response to acute seizures induced by chemoconvulsants and electrical stimulation includes a rapid activation of glia, enhancement of inflammatory cytokines consisting of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF-a) in microglia and astrocytes, and is followed by a cascade of downstream inflammatory events which include the activation of TLR, NF-κB, complement system, chemokines and acute phase proteins. Glia activation and cytokines up-regulation may promote the origins of epilepsy, and contribute to seizure-related hippocampal pathology, such as neuron death, reactive gliosis, and mossy fiber sprouting.Data in vivo and in vitro revealed that the neuroprotection of estrogen in brain trauma, stroke and neurodegenerative diseases was mediated by inflammatory inhibition. As there has no accepted definition about the dose of administration of estrogen and the larger dose commonly possesses unpredictable side effects, we choose 10μg/rat as our dosage. Our experiment is aimed to study the effects of low dose EB on the hippocampal neurodegeneration and the related inflammatory process as well as the expression of estrogen receptor in the pilocarpine-induced epileptic rats. PartⅠEffects of low dose estrogen on the behaviors and hippocampal neurodegeneration of the epileptic ovariectomized female ratsObjective:To observe the effects of low dose estrogen on the behaviors and hippocampal neurodegeneration of the ovariectomized female rats after Pilo-SE.Methods:Adult female SD rats with at least two normal 4-day estrous cycles were used for ovariectomy. After 7 days, rats were injected subcutaneously with either 17β-estrodiol benzoate (EB; 10μg/0.1ml) or sesame oil (0.1ml) for 4 consecutive days. 6 h after the last dose, the rats were injected intraperitoneally with pilocarpine (320mg/kg) or saline at the same volume. Latencies to first seizure and severe seizures (4/5 degree) were recorded as well as mortality. Only rats with continuous long-lasting behavioral seizures (SE) were employed. Animals were injected with 5mg/kg diazepam intraperitoneally 1 h after the onset of SE and were perfused and decapitated separately at 8 h or 24 h post-SE. Blood samples were collected for detecting the level of serum estradiol with methods of electrochemical illusion and brains were fetched and made into frozen slices. Toluidine blue staining was utilized to determine the morphological and distributing characteristics of neurons in hippocampus. Immunofluorescence methods were adopted to assess the expression of Fluoro-Jade B and NeuN IR positive cells. Results:1. Within the 35 rats injected with pilocarpine,31 were successfully ilicited SE. Low dose of EB had no influence on the latencies to the first seizure and severe seizure onset. Survival rates were similar between the two groups of 8 h or 24 h post-SE.2. The serum estrodiol levels at 24 h post-SE were significantly enhanced in the EB treatment groups compared with the non-EB treatment groups (P＜0.01), and approximately 2.5 times more than the levels of the proestrus (122.8±9.19 pmol/L). Seizures induced by pilocarpine had no impact on the serum estrodiol levels.3. Toluidine blue staining indicated that the epileptic rats manifested with intact pyramidal cells in hippocampus as in the non-epileptic rats at 8 h post-SE, but reveals significant neuronal cell damages in areas CA1, CA3 and hilus when 24 h after SE onset. Low dose of EB preserved the neuronal population in the hilus at the 24 h post-SE, but had no effect on neuronal survival of areas CA1 and CA3.4. Immunofluorescence staining of NeuN performed at 24 h post-SE revealed a reduction of neuronal staining in all subfields of hippocampus in the epileptic group, and therapeutic injection of EB promoted a remarkable neuroprotective effects on vulnerable hilar neurons but no marked benefit to the areas CA1 and CA3.Conclusions:Low dose of EB (10μg/rat) injected subcutaneously for 4 consecutive days raised the levels of serum E2 up to 2.5 times more than that of proestrus. The serum E2 levels were not changed by pilocarpine-induced seizures. Low dose of EB had no significant influence on the latencies to the first seizure, severe seizures and survival rate. Status epilepticus induced by pilocarpine could result in the delayed neuronal cell loss (24 h post-SE) in the subfields of hippocampus when maintained for 1 h, and low dose of EB pretreatment presented remarkably neuroprotection in the hilus.Part IIEffects of low dose estrogen on the hippocampal inflammation of the epileptic ovariectomized female ratsObjective:To observe whether inflammation participates in the neuroprotection of EB in the Pilo-SE. Methods:Animal models were made according to the first part and decapitated at 8 h or 24 h after SE. The bilateral hippocampi were completely peeled off and blood samples were collected. The right hippocampi were homogenated for the supernatant and blood were centrifugalized for serum. IL-1βand TNF-a levels were detected in the supernatant and serum with the method of enzyme-linked immunosorbent assay. Brain slices of all rats were prepared for CD11b and NeuN immunofluorescence staining to observe the microglia and astrocytes in the hippocampus. Results:Levels of IL-1βand TNF-a in the hippocampus were remarkably elevated at 8 h after seizures induced by pilocarpine and declined back to the control levels at 24 h. Levels of IL-1βand TNF-a in the pilocarpine group were approximately 226% and 261% compared to the control group at 8 h post-SE. The Pilo+EB group had lower IL-1βlevel in the hippocampus than the pilocarpine group (P＜0.05), but the TNF-a level were parallel between the two groups. No significant influences were found by the seizures or low dose of EB pretreatment on the IL-1βand TNF-a serum levels. Microglia and astrocytes were activated by seizures as early as 8 h after SE, and were much more evident at 24 h. Activated glia manifested with large cell body and thick ramification. Some microglia showed "ameboid type". The number of activated microglia was significantly reduced in the hilus of the Pilo+EB group compared with pilocarpine group at 8 h or 24 h after SE (P＜0.05). The CA3 area of Pilo+EB group had less activated astrocytes than the pilocarpine group (P<0.05), but with no difference in the CA1 or hilus areas at 24h post-SE. Both groups had similar number of activated astrocytes at 8 h post-SE. Conclusions: Seizures induced by pilocarpine could contribute to the activation of microglia and astrocytes, up-regulation of IL-1βand TNF-a in the hippocampus, but bring no effect to the serum levels of IL-1βand TNF-a. Low dose of EB pretreatment could reduce the expression of IL-1βin the hippocampus, decrease the activation of microglia in the hilus area and diminish the activation of astrocytes in the CA3 area. Low dose of EB could inhibit inflammation in the hippocampus of the pilocarpine-induced epileptic rats.Part IIIEffects of low dose estrogen on the hippocampal estrogen receptor of the epileptic ovariectomized female ratsObjective:To observe the effects of low dose EB on the expression of hippocampal estrogen receptors in the epileptic ovariectomized female rats. Methods:The left hippocampi of the epileptic rats, which were sifted in the second part, were homogenated for the supernatant and Western Blot was utilized to detect the expression of ERa and ERβ. Results:The ERa expression in the hippocampal supernatant remained the same among different groups at 8 h or 24 h post-SE. Pretreatment with low dose of EB and seizues induced by pilocarpine exerted little impact to the expression of ERa in hippocampus. The hippocampal ERP expression showed no differernce among 8 h groups after SE, but it was signigicantly reduced in the Pilo+EB group compared with the EB (P＜0.01) and pilocarpine groups (P＜0.05) 24 h post-SE. Conclusions:The expressions of ERa and ERβwere not influenced by seizures induced by pilocarpine. Low dose of EB pretreatment demonstrated no effect to the hippocampal ERa and ERP expression in the non-epileptic rats at 8 h and 24 h post-SE, but it significantly reduced the ERP expression in the hippocampus of the epileptic rats at 24 h post-SE. 1. Low dose of EB (10μg/rat) injected subcutaneously for 4 consecutive days could maintain the serum E2 at a high level. It is approximately 2.5-3 times more than the levels of the proestrus 30 hours above after the last injection. Serum E2 levels were not markedly influenced by seizures.2. Low dose of EB had no obvious impacts on the behavior of epileptic rats, for example, the latencies and mortalities, but it could significantly reduce the neurodegeneration in hilus area at 24 h after SE.3. Pilocarpine-induced seizures could contribute to the activation of glia, up-regulation of IL-1(3 and TNF-a in the hippocampus, but had no effect to the serum IL-1βand TNF-a level. Low dose of EB pretreatment could reduce the expression of IL-1βin the hippocampus, decrease the activation of microglia in the hilus area and prohibit the activation of astrocyte in the CA3 area.4. Glia activation and inflammatory cytokines synthesis precede the appearance of neuronal damage suggesting its association with the appearance of cell injury. Low dose of EB might exert neuroprotective effects via the reduction of the inflammatory process in hippocampus. The effect of EB might be related to the in hippocampus, and little influences of ERa had been detected in this study.