Activation Of ERK Pathway By Spontaneous Seizures And Its Effects On Neurogensis In A Mouse Model Of Epilepsy

Part one:A mouse model of temporal lobe epilepsy characterized with spontaneous seizures and its influence on adult neurogenesisObjective:To investigate a useful pilocarpine model of temporal lobe epilepsy (TLE) characterized by recurrent spontaneous seizures, and to explore its influence on the adult neurogenesis of hippocampal formation.Methods:1.Young adult (6-8 week of age) C57BL/6 male mice were used in the study. Sustained seizures were induced in experimental animals by the administration of pilocarpine. After recovery from status epilepticus (SE), the behaviors of pilocarpine-treated mice were continuously monitored for 50 days (n=8).Then the mice were stained with cresyl violet for the historical study.2.DCX, a neural progenitor cell marker, was detected by immunohistochemistry and Western blot method at 1 week,2 weeks,4 weeks,8weeks,12 weeks after SE, as well as in control animals (n=3, respectively).Results:1.Fifty-day behavior study showed that 87.5% of pilocarpine-treated mice developed spontaneous seizures.They usually experienced a latent period from 3-14 days after SE before they begin to have spontaneous seizures.The duration ranged from 10-40 seconds. Spontaneous seizures occurred 2.18±0.45 per week on average, usually during the light on/off period and appeared in clusters.2.The Nissl staining of the pilocarpine treated mice consistently showed hilus cells loss, and usually accompanied by cell loss in CA1 area and disorganization of CA1,CA3 pyramidal cell layers.In addition, some animals also showed cell loss in dentate granule cell layer or granule cell layer dispersion.3.The Western bolt results revealed that the level of DCX began to increase at 1 week after SE in the hippocampus formation, and downregulated to basal level at 8 weeks after SE. The immunohistochemistry of DCX showed that the DCX-labeled cells were located in the subgranular zone (SGZ).After SE, they were originally appeared in clusters, and then immigrated to dentate inner granule cell layer and occasionally to hilus or outer layer of dente gurus.Conclusions:The mouse model of TLE with spontaneous seizures shares most of the characteristics of TLE patients, meanwhile it showed consistently increased neurogenesis which could also be observed in TLE patients.It offered a useful tool to help investigate the ongoing epilepsy-related processes associated with spontaneous seizures. Part two:Activation of ERK pathway by spontaneous seizures and its potential effects on neurogenesis in a mouse model of TLEObjective:To evaluate the dynamic changes of ERK pathway in the mouse model of epilepsy with spontaneous seizures,and to explore the influence of ERK pathway activated by spontaneous seizures on neurogenesis.Methods:1.Pilocarpine-treated mice from 2 to 7 weeks following SE were monitored for spontaneous seizures, and a total of 17 pilocarpine-treated animals were included in the pERK study. The mice were purfused at different intervals following a spontaneous seizures, including 2min (n=6),3min (n=3),6min (n=2),30min (n=3)following seizure detection. Animals that demonstrated additional spontaneous seizures during the previous 12-24h before perfusion were excluded. For comparison, age-matched epileptic mice (n=3)following SE that had not experienced a behavioral seizure in the last 24h prior to perfusion were included. Age-matched control animals were included in the study and were perfused at the same time as the pilocarpine-treated animals.In addition, for comparison of pERK labeling, the immunohistochemistry of Fos and GABAARδsubunit were also studied at the onset of a spontaneous seizure (2 min).2.In the pilocarpine-treated animals which experienced a spontaneous seizure at 2 min before perfusion, several neural progenitor cell (NPC) markers were used for double immunofluorescence labeling with pERK in the hippocampal formation:(1)radial glia-like NPC markers, including GFAP, BLBP, Nestin and Pax6; (2) a marker of intermediate stage NPCs with neuronal lineage potential, NeuroD; (3) immature neuronal markers, including DCX, Tuj-1;(4) an early granule cell-specific marker, Prox1;(5)a general neuronal marker, NeuN; (6) A marker of mature astrocytes,S100β.In addition, proliferation markers, Ki67 and Mcm2 were also used to determine if pERK labeled cells were in the proliferative cycle.3.To evaluate the difference of Mcm2 labeled cells and the BLBP-expressing NPCs that were in the proliferateive cycle in the SGZ between pilocarpine-treated animals at the onset of a spontaneous seizure and control animals, the density of Mcm2 labeled cells and the percentage of double-labeled cells that expressed BLBP and Mcm2 were calculated (n=5).4.Triple immunofluorescece of pERK, BLBP and Mcm2 were conducted in the hippocampal formation of pilocarpine-treated animals at the onset of a spontaneous seizure, and the percentages of single-, double-and triple-labeled cells were calculated (n=3).Results:1.In the control animals, the pERK-labeled cells were scattered in the dentate gyrus.In the pilocarpine-treated animals, when the mice had not experience spontaneous seizure in the last 24h before perfusion, the pERK labeling was obviously decreased. At the onset of a spontaneous seizure, the pERK labeling dramatically increased, it first appeared in the cells located in the SGZ at 2 min after a spontaneous seizures, and quickly spread to the whole granule cell layer, then went back to basic level within 30min. For comparison of pERK labeling with Fos and GABAARδsubunit, The Fos labeling were relatively delayed compared to pERK staining although they shared the similar changes; The area with pERK upregulation showed downregulation of GABAARδsubunit expression.2.In the animals that just experienced a spontaneous seizure at 2 min before perfusion,77.62±4.89% of pERK labeled cells colocalized with BLBP,77.68±5.50% of pERK labeled cells colocalized with GFAP, 21.67±7.12% of pERK labeled cells colocalized with NeuroD, and less than 5% of pERK labeled cells meanwhile expressed PSA-NCAM. Virtually no expression of DCX, Tuj-1, Prox1 or NeuN was found in pERK labeled cells.3.In the animals that just experienced a spontaneous seizure at 2 min before perfusion, the density of Mcm2 labeled cells were increased compared to that in the control animals. Similarly, the potential of BLBP labeled cells that were proliferating was also increased.4.In the animals that just experienced a spontaneous seizure at 2 min before perfusion, virtually all BLBP and Mcm2 double-labeled cells meanwhile showed ERK activation.Conclusions:1.ERK pathway activation showed dynamic changes following spontaneous seizures, which suggests it could be a sensitive marker considering the spontaneous seizure activity.2.In the pilocarpine treated animals with spontaneous seizures, the proliferative potential of neural stem cells that located in the SGZ were increased.3.At the onset of a spontaneous seizure, ERK pathway activation was first appeared in the neural stem cells and the NPCs with neuronal potential.4.Spontaneous seizure could exert its influence on the proliferation of NPCs and may contribute to the epilepsy-related neurogenesis.