| Part One Mouse pilocarpine model of temporal lobe epilepsy and time course changes of neuron and astrocyte in epileptic hippocampusObjective To investigate time course changes of neuron and astrocyte in epileptic hippocampus of pilocarpine model of temporal lobe epilepsy.Method6-8weeks old adult C57BL/6male mice were randomly divided into experimental group and control group. In experimental group, status epilepticus (SE) was induced by intraperitoneal injection of pilocarpine, while the controls were injected with equal dose of saline. After SE, behavior of mice was observed and spontaneous seizures were recorded. Immunohistochemistry of NeuN and GFAP were used to evaluate the time course changes of neuron loss and gliosis2h,24h,7d,15d,60d after SE.Results1. The successful rate of mice model of temporal lobe epilepsy was48.0%and total mortality rate was40.3%.87.5%of model mice developed SS, and the average latent period was11.1±4.8days. SS were often observed during the light on/off period and lasted for10～40seconds, usually less than1minute.2. Immunohistochemistry of NeuN:Neuron loss occurred in in area CA1-3and hilus area after SE:Neuron loss of CA1area occurred24h after SE, while hilus cells loss can be observed as early as2h after SE, the cells loss of these two area can be consistently; CA3area can show neuron loss in the early time and chronic stage; granule cells in DG area increased7d after SE, some epileptic mice also showed granule cell layer dispersion.3. Immunohistochemistry of GFAP:Compared to neuron, the morphological changes of astrocyte occurred later. The cells showed a edematous appearance24h after SE, the positive staining area in CA1, CA3and hilar increased (P<0.05); the most remarkbly gliosis was observed7d after SE, which declined in chronic stage.Conclusion1. Pilocarpine mouse model of temporal lobe epilepsy showed typical pathology of neuron loss and gliosis2. The pilocarpine model of C57BL/6mice is an ideal temporal lobe epilepsy model.Part Two Altered expression of Pannexin protein in hippocampus of mouse model of temporal lobe epilepsyObjective To investigate expression of Pannexin protein in hippocampus of mouse model of temporal lobe epilepsyMethod6-8weeks old adult C57BL/6male mice were randomly divided into experimental group and control group. In experimental group, status epilepticus (SE) was induced by intraperitoneal injection of pilocarpine, while the controls were injected with equal dose of saline. Immunohistochemistry of Panxl and Panx2were used to evaluate the expression of Panx in epileptic hippocampus2h,24h,7d,15d,60d after SE. Moreover, NeuN and GFAP were used for double immunofluorescence labeling with Panxs in the hippocampal formation.Results1. Panxl and Panx2immunohistochemistry:In hippocampus, both Panxl and Panx2were expressed in the entire CA region, the granule cells of the dentate gyrus and in individual neurons in the stratum oriens and stratum radiatum. After SE, the expression of Panxl and Panx2in principal cells decreased; while increased expression of Panxl could be observed in non-principal cell area, especially in stratum radiatum.2. Double immunofluorescence labeling of Panxs and NeuN:All hippocampal Panx-positive cells were confirmed to be neurons because of double-labelling with NeuN in control group. Double-labelling cells of pyramidal layer were at the lowest lever7d after SE, the cells loss of hilar area can be consistently.3. Double immunofluorescence labeling of Panxs and GFAP:In control group, GFAP labelled cells didn’t show Panxl and Panx2positivity. Double labelled Panxl and GFAP could be observed in the cells particularly locationed in stratum oriens and stratum radiatum of CA1and CA3area after SE, which was most notable7d or15d after SE. During the whole course, there was no evidence of double labelled Panx2and GFAP.Conclusion1. Altered expression of Panxl and Panx2protein can be detected in hippocampus of pilocarpine model of temporal lobe epilepsy.2. Panxl abundantly expressed in astrocyte after SE.Part Three The role of Pannexin channels in regulating pilocarpine-induced seizures and hippocampal neuron-glia plasticityObjective To explore the effect of Panx channel on pilocarpine induced seizures in vivo and the hippocampal neuron-glia plasticity after SE.Method6-8weeks old adult C57BL/6male mice were randomly divided into three groups:10Panx group, Cbx group and ACSF control group. Mice of each group were treated respectively with intracerebroventricular injection of10Panx, Cbx and ACSF. About5hours after injection, all the mice received the examination of seizure susceptibility:after intraperitoneal injection of pilocarpine, mice were observed for2hours for the emergence of first seizure attack (stage≥3), the proportion of seizures of each grade and the mortality rate. Only those mice up to the SE standard were prepared for next NeuN and GFAP immunohistochemistry60d after SE.Results1. The results of seizures susceptibility:the latency to the first stage3seizure in10Panx group and Cbx group were71.40±37.33min and59.53±34.69min, which were significantly longer than control group (P<0.01). The ratio of severe seizures (stage≥5) in control group was50%, but only20%and13.3%in10Panx group and Cbx group. The mortality rates within2hours were20%(10Panx group),13%(Cbx group) and40%(ACSF group).2. Immunohistochemistry of NeuN:Neuron loss can be observed in CA1-CA3area and hilar in the hippocampus of mice from every group. Compared with ACSF control group, loss of cells were softer in CA1and CA3pyramidal layer of10Panx and Cbx group, together with Hilar area of10Panx group (P<0.05); the difference between DG area of three group was not significant.2. Immunohistochemistry of GFAP:Morphological changes of astrocyte were detected all of the three groups. Compared to control group, gliosis in CA1area of10Panx and Cbx group and DG area in Cbx group was not as serious (P<0.05); there was not significant difference of gliosis between experimental and control group in other areas of hippocampus.Conclusion 1. Block of Panx channels inhibited pilocarpine induced seisures in vivo.2. Panx channels may play a role in regulating hippocampal neuron-glia plasticity after SE... |
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