Preliminary Study On The Mechanism Of Microglia In Epilepsy In Mice

Objectives:In this study,pilocarpine-induced epilepsy model was established to explore the effects of microglia activation on acute and chronic inflammation in epilepsy mice,and to explore the possible mechanism.Methods:The epilepsy model induced by pilocarpine was intervened with minocycline,an inhibitor of microglia activation,and 4-aminobenzoylhydrazide,a specific inhibitor of myeloperoxidase.Healthy male C57BL/6 mice were divided randomly into ABAH treatment group,minocycline treatment group,saline treatment group and sham group.The experiments were divided into two parts.In the first part,the experiment was terminated 24 hours after the epilepsy model was induced successfully,then the acute phase of seizures were collected.The expression of inflammatory factors in serum of each group was measured by ELISA.The expression of MPO,P-p38,TNF-? and IL-10 in brain tissues was detected by Western blotting.The microglial activation and MPO were observed through immunohistochemical staining and immunofluorescence coexpression.In the second part,after the epilepsy model was induced successfully,the experiment was terminated after 60 days of intervention treatment,thus the specimen of spontaneous recurrent epilepsy at chronic stage were collected.Except for the indicators in the first part,the number of spontaneous recurrences of epilepsy mice was recorded from the second day to the last day of the experiment.Results:Part ?: 1.Model induction results: 30 mice were induced to epilepsy model,23 were induced successfully and 7 died.The success rate of induction was 76.7%.2.ELISA results have showed that the expression of TNF-? and IL-10 in serum of epilepsy mice increased significantly.The expression of TNF-? reduced significantly after the treatment with ABAH and minocycline,and the expression of IL-10 increased significantly.3.Western blotting: The expression of MPO,P-p38,TNF-? and IL-10 in the brain tissues of the saline treatment group increased.After the treatment with ABAH and minocycline,the expression of MPO,P-p38 and TNF-? reduced,and the expression of IL-10 increased significantly.Immunohistochemical results suggested that the number of Iba1+ cells in the brain tissue of the saline-treated group increased significantly,and the expression of MPO also increased.After treatment with ABAH and minocycline,the number of Iba1+ cells in the brain tissues of epilepsy mice decreased significantly,while the expression of MPO decreased.In immunofluorescence staining,compared with the sham group,the number of MPO+ and Iba1+ cells in the hippocampus of the saline-treated group was increased obviously.Moreover,the number of MPO+ and Iba1+ cells in the ABAH-treated group and the minocycline-treated group reduced significantly compared with the saline-treated group.And MPO and Iba1 were co-expressed.Part ?: 1.Model induction results: 30 mice were induced for experiments,22 were induced successfully and 8 died.The success rate of induction was 73.3%.2.Behavioral observation results: No seizure was observed in all animals in the sham group.Epilepsy recurrences were observed 4 times of 6 mice in the saline treatment group,1 time of 8 mice in the minocycline treatment group,and no epilepsy recurrence was observed in 8 mice in the ABAH treatment group.3.ELISA results indicated that the expression of TNF-? and IL-10 in serum increased significantly in the saline treatment group.The expression of TNF-? reduced significantly after treatment with ABAH and minocycline,and the expression of IL-10 increased significantly.4.Western blotting: The expression of MPO,P-p38,TNF-? and IL-10 in brain tissues of the saline-treated group increased.After the treatment with ABAH and minocycline,the expression of MPO,P-p38 and TNF-? reduced,and the expression of IL-10 increased significantly.5.The results of immunohistochemistry illusrated that the number of Iba1+ cells and the expression of MPO in the brain tissues of the saline treatment group was quite different from the sham group.The number of Iba1+ cells in the brain tissues of epilepsy mice reduced slightly after the treatment with ABAH and minocycline.The expression of MPO was also reduced slightly.6.Immunofluorescence labeling: Compared with the sham group,the number of MPO+ and Iba1+ cells in the hippocampus area of the saline treatment group increased slightly.Compared with the saline treatment group,the MPO+ and Iba1+ cells in the ABAH treatment group and the minocycline treatment group increased slightly.Conclusions:1.The number of microglial cells activation increased significantly in the acute phase of epilepsy mice.2.The activation of p38/MAPK signal pathway and the expression of MPO and inflammatory factors increased during the pathogenesis of epilepsy mice.3.To up-regulates the expression of MPO by the p38/MAPK pathway,microglial activation may promote the inflammatory response in epilepsy mice.Inhibiting microglial activation,the epilepsy recurrence in mice reduced.