Effects Of STAT3 On Reactive Astrocytes Following Pilocarpine-Induced Rat Seizures

Objective: To investigate the spatiotemporal distribution pattern of phospho-STAT3 (p-STAT3) and reactive astrocytes in rats hippocampus following pilocarpine-induced seizures,and to infer the role of STAT3 signaling pathway in gliosis of rats with epilepsy.Methods: Rat temporal lobe epilepsy models were established by intraperitoneal injection of pilocarpine (PILO) , and specific STAT3 inhibitor-AG490 was used to set up pretreated models,saline was insteaded in the contral group. The expression pattern of p-STAT3 and glial fibrillary acidic protein (GFAP)-positive cells was observed immunohistochemically before and after blockage of the JAK/STAT pathway in rat hippocampus. Double immunofluorescence assay was used to investigate the relationship between p-STAT3 and GFAP-positive cells.Results:1. Ethology observation: No seizures were observed in the saline-treated group.Among the pilocarpine-treated rats, 85.00% (34/40) rats presented seizures more than III stage (Racine classifacation),7.50% (3/40) rats died, and 7.50% (3/40) rats were expelled because of no epileptic attack arrived to III stage; in this group, the rate of spontaneous recurrent seizure (SRS) is 4.52±1.49 times per rat. Among the AG490-pretreated rats, 82.50% (33/40) rats presented seizures above III stage, 15% (7/40) rats died and 2.50% were expelled, while SRS rate (1.26±0.78 times per rat ) decreased remarkably compared with pilocarpine-treated groups.2. Histopathology observation: Neurons of hippocampus had no marked changes in saline-treated rats. While in pilocarpine-treated group, neuronal degeneration,necrosis and slightly cell loss can be observed in the CA1,CA3 and hilar region of hippocampus since 3 hours after SE. These patho-changes became most serious at 3 days after SE. Granular cells in the dentate gyrus (DG) preserved integrited cell structure at 3h while showed degeneration at 3d. After 15d necrosis of neurons lessened gradually. Compared with the pilocarpine-treated group, necrosis of neurons lessened in the AG490-pretreated rats.3. Immunohistochemistry Results: Few pale-staining p-STAT3 kytoplasm- positive cells were observed in the hippocampus of saline-treated rats; Among pilocarpine-treated group, the p-STAT3 immunoreactivity was main observed in necleus, and the numerus of these p-STAT3 positive cells increased at 3h after seizure, reached peak at 3d, then graudually decreased, and kept at a higer level than the basic till 30d. All these immunoreactivity was especially strong in the CA1,CA3 and hilar regions of hippocampus. Compared with the pilocarpine-treated group, the immunoreactivity of p-STAT3 in AG490-pretreated rats was significantly blocked. The GFAP immunoreactivity was observed only in few cells in the contral group, with which have little cell body and tiny arborization. Among pilocarpine-treated group, the section area of GFAP-positive cells enlarged with its evection increased, and the distribution pattern of GFAP positive cells was smilar to the p-STAT3 expression. Compared with the pilocarepine-treated group, the immunoreactivity of GFAP in AG490-pretreated rats weakened obviously at most time course.4. Double Immunoflourescence Assay: The GFAP positive cells were stained with green astro-kytoplasm, and the p-STAT3 positive cells were with red round or oval-shap necleus. Both of these two positive cells were main distributed in the CA1 and hilar regions of hippocampus. The p-STAT3 positive cells with red nucleus can also be observed in GFAP positive cells'kytoplasm, revealed that the GFAP immunoreactive astrocytes may accompany the activation of STAT3.Conclusion: Rats model of chornic epilepsy induced by Lithium-pilocarpine can mimic features of human temporal epilepsy in seizure behaviors and histopathology findings. Gliosis in hippocampus was adaptive reaction to seizures and neuron necrosis, it might play an important role in recurrence of epilepsy. The STAT3 signaling pathway can be activated in reactive astrocytes of rats with seizures induced by pilocarpine, indicating that the phosphorylation of STAT3 might contribute to the reactive gliosis. AG490 could block the activation of STAT3, and also can effect the seizure behaviors and the histopathology changes as well as the reactive gliosis, indicating that it might have a negtive role in the epilpsy attacks.