| Objective(1)To observe the expression of cytochrome P450 aromatase within neurons and astrocytes in the hippocampus of female rats with different estrous cycle.(2)To observe the changes of P450arom expression within neurons and astrocytes in the hippocampus after the onset of seizures,define the relationship to the severity of seizures,and the source of brain endogenous estradiol.Methods(1)Adult Sprague-Dawley rats(weight240～260g) were divided into 6 groups: proestrus group,estrus group,metestrus group,diestrus group,ovariectomized group of female rats and male group,each group has 6 rats.After perfused with 4℃saline and 4%paraformaldehyde solution,the brains were harvested after decapitation. These brains were fixed with 4%paraformaldehyde solution for 12h and dehydrated with 10%,20%,30%saccharose gradient.Frozen sections were prepared for staining. (2)The female SD rats were raised for two weeks after ovariectomy (weight280～300g),and randomly divided into 12 groups,including 6 KA groups and 6 NS control groups.On the 15th day,the rats were anesthetized and fixed to the stereotaxic apparatus,1μg dose of KA were injected into the right amygdale slowly to induce the complex partial seizure(5μl NS was injected into the right amygdale of the NS group rats).The latency to severe seizure stage,the frequency and duration of the seizure were recorded.The samples were prepared according to the protocals described in part one after 1h,2h,3h,4h,5h,6h after seizure were collected respectively.(3)Dual immunofluorescence staining were used to measure the positive cells of aromatase in neurons and astrocytes:a.Neurons and astrocytes were marked with NeuN and GFAP antibodies.b.Aromatase was marked with aromatase polyclonal antibody.c.Fluorescence microscope and Laser confocal microscope were selected for the staining results.Results(1)The results of immunofluorescence showed the P450arom immunoreactive cells distributed in the CA1 to CA3 and DG region of hippocampus,maily in CA1 and DG region.They expressed on both membrane and nuclea of neuron.(2)A large quantity of P450arom immunoreactive cells were found in the estrus group.In CA1 and DG regions,there were statistically significant difference with other groups of female rats. The P450arom expression of ovariectomized group in hippocampus were obviously weaker than normal female rats(p＜0.05).(3)Statistically significant difference were also found between male group and female group,while male group had a strong expression.(4)The P450arom still maily expressed in neuron after seizure onset,and the quantity of immunoreactive cells didn't have a statistically significant change during the stage of 1-6 hours after seizure onset.(5)At the 5th and 6th hours after seizure onset,the expression of P450arom appeared on astrocyte.The the quantity of immunoreactive cells coexpression with GFAP had a statistically significant difference compared with the 0-4 hours after seizure onset and the NS groups of the same time point.(6)Approximately 20-30min after KA intra-amgydale injection,the rats began to show mild seizure.The peak were reached in 180 min after KA injection and then the seizure was alleviated.There were no correlation between seizure severity scale and quanitity of P450arom immunoreactive cells.ConclusionsThe P450arom expression could be detected in hippocampus and maily localized in neuron,which indicated estradiol can be synthetized locally in hippocampus of neurons.(2)There were dynamic changes of the quanitity of P450arom expression during estrous cycle.(3)There were sex difference of P450arom expression in rats.(4) The expression of P450arom were still maily localized in neuron after seizure onset, however there were a small quanitity of P450arom expression appeared on astrocyte. (5)The quantity of P450arom in neuron didn't have a significant change after seizure onset.(6)The quantity of P450arom in astrocyte obviously increased in telophase of epileptic paroxysm,which indicated estradiol can also be synthetized by astrocyte in seizure rats hippocampus.
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