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Objective:To investigate the effects of hippocampal injection of tyrosine kinase receptor binding protein B3(Ephrin-B3) agonist on spontaneous seizures and the expression of hippocampal secretory glycoprotein (Reelin) and phosphorylated adaptor protein (p-Dab1) in epileptic model rats.Methods:Seventy-eight rats were randomly divided into control group and model group according to body mass matching with 39 rats in each group.The rats in control group were fed normaly, and the rats in model group were established epilepsy model by intraperitoneal injection of lithium chloride pilocarpine. The hippocampal tissues were taken in the acute phase (7 days), quiescent phase (14 days) and chronic phase (60 days) after the successful induction of status epilepticus. The levels of Reelin protein and p-Dab1 protein in the hippocampal tissues of epileptic model rats and normal rats were detected by immunohistochemistry and Western blot.And thirteen rats were randomly selected at each time point. Another 48 rats were randomly divided into normal Fc-control group, normal EphB3-Fc group, epilepsy Fc-control group and epilepsy EphB3-Fc group, with 12 rats in each group. Rats in the first two groups were fed normally, and those in the latter two groups were established epileptic model. Seven days after modeling, all rats were injected into bilateral hippocampus with EphB3-Fc (Ephrin-B3 agonist) and FC control (control agent of Ephrin-B3 agonist) according to the grouping, once a day for 7 days. After administration, the changes of behavior and EEG were observed within two weeks. At the same time, the expression of Reelin protein and p-Dab1 protein were detected by immunohistochemistry and Western blot. SPSS 21.0 was used for statistical analysis, One-way ANOVA was used for multi group comparison, and Tukey's test was used for pairwise comparison.Results:The results of immunohistochemistry and Western blot showed that compared with the control group, the levels of Reelin and p-Dab1 protein in hippocampus of model group decreased significantly at 7, 14 and 60 days after epilepsy (all P<0.01). The results of immunohistochemistry showed that compared with epilepsy Fc-control group, the levels of p-Dab1 ((0.41±0.04), (0.58±0.06), P<0.05) in epilepsy EphB3-Fc group increased significantly.Western blot result showed that the level of p-Dab1 in epilepsy EphB3-Fc group increased compared with that of epilepsy Fc-control group (1.34±0.04), (2.26±0.10), P<0.01). Compared with epilepsy Fc-control group, epilepsy EphB3-Fc group showed less average seizure duration ((39.00±1.79)s, (26.50±1.87)s; t=23.21, P<0.01), less frequencies ((2.00±0.89), (0.50±0.55); t=2.32, P<0.01) and less latent period ((6.33±1.37)day, (12.50±1.87)day; t=2.52, P<0.01) in spontaneous recurrent seizures. Compared with epilepsy Fc-control group, epilepsy EphB3-Fc group showed lower average amplitude ((37.30±1.21)μV, (29.00±1.41)μV; t=25.14, P<0.01), less average seizure duration ((5.35±0.19)s, (2.35±0.19)s; t=3.13, P<0.01). Conclusion:Ephrin-B3 alleviated spontaneous recurrent seizures by upregulating Reelin and p-Dab1 in temporal lobe epilepsy rat.
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@#Objective To explore dynamic expression of Ephrin-B3 in hippocampus of pilocarpine induced epileptic rats and the role of Ephrin-B3 in the process of epileptogenesis.Methods We first established pilocarpine induced epileptic rat model and then detected Ephrin-B3 expression at 7,14 and 60 days post status epilepticus (SE) by real time qPCR,immunohistochemistry and Western blot.Results After pilocarpine injection,rats showed facial muscles,forelimb and tail clonus and ataxic lurching,head bobbing and wet dog shakes.Real time qPCR revealed that Ephrin-B3 mRNA expression in hippocampus decreased during spontaneous seizure period.Compared with controls,epileptic rats showed decreased Ephrin-B3 expression at 7 days,reached the lowest level at 14 days and then increased slightly at 60 days post-SE (P<0.001).Ephrin-B3 protein in hippocampus also decreased at 7,14 and 60 days detected by Western blot (P<0.001).Immunohistochemistry results showed that Ephrin-B3 mainly expressed in granule cells in dentate gyrus.Analysis of immunohistochemistry revealed decreased Ephrin-B3 expression in epileptic rats compared with controls (P<0.001).Conclusion The dynamic changes of Ephrin-B3 expression in hippocampus of epileptic rats indicated that Ephrin-B3 may participate in the process of spontaneous recurrent seizures.
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Objective To explore the expression of drug transporter organic anion transporting polypeptide2(Oatp2) and P-glycoprotein(P-gp) in brains of chronic epileptic rats induced by lithium-pilocarpine.Methods Chronic epilepsy modles were elicited after status epilepticus(SE) induced by lithium-pilocarpine in adult Wistar rats.The expressions of Oatp2 and P-gp were detected by immuno-histochemistry(SABC) method.Results Positive expression of Oatp2 was predominantly in the plexus epithelial cells and(blood-brain) barrier endothelial cells.Weak staining for Oatp2 was also detected in the neurocyte.In contrast,apparent expression of(P-gp) was seen in brain capillary and neurocyte only.Compared with normal control rats,expression of Oatp2 on the plexus epithelial cells and brain capillary in chronic epileptic rats was significantly decreased(P